News Article | May 3, 2017
The Analyses and Estimates the CRISPR Market by: Type, End-Users, Geography SA-BRC is pleased to announce the initiation of research on "Global CRISPR Products and Services Market." Houston, TX, May 03, 2017 --( Rapid growth in biotechnology and advances in genetic engineering has accelerated life science research. Increasing understanding of genetic makeup of various life forms and their role has provided research across the globe with solid grounds to discover novel therapeutic and commercially viable alternatives. With current alarming rise in incidence of chronic diseases such as cancer, diabetes, and various other cardiovascular and neurological disorders, there is an urgent need of long term treatment substitute. Advances in genetic research such as the CRISPR technology would help in achieving these milestones earlier. Request Free Report Sample@ www.sa-brc.com/Global-CRISPR-Products-and-Services-Market-Assessment--Forecast-2017-2021/up100 Although low cost, and ease of use are beneficial for genetic research, scientists also believe that widespread and uncontrolled sale of such genetic engineering tools through e-commerce websites with increase the risk of bio-hacking. The CRISPR kits are available online at a price of less than US$ 200. This encourages uncontrolled and unauthorized use of biotechnology tools in modifying genetic sequences. Such research activities are likely to result in new disease causing agents that can affect large percentage of population globally. Various genetic researchers, advocacy groups and industry experts have demanded control of regulatory authorities for sale of CRISPR kits over e-commerce websites. Implementation of restraints on online sales would drastically affect the CRISPR market growth. Companies are seen investing heavily in providing cost efficient and CRISPR kits for more accurate results. In 2017, Synthego raised US$ 41 million to fortify its portfolio for CRISPR gene editing kits. Key players in the clustered regularly interspaced short palindromic repeats (CRISPR) market include ODIN, Thermo Fisher Scientific, Inc., OriGene Technologies, Inc., Cellecta, Inc., Takara Bio, System Biosciences, Inc., BioCat GmbH, Synthego Corporation, QIAGEN, Biolegio B.V. and various others. Request For TOC@ www.sa-brc.com/Global-CRISPR-Products--Services-Market-Assessment--Forecast-2017-2021/upcomingdetail100 Houston, TX, May 03, 2017 --( PR.com )-- Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) are partially palindromic repeated DNA sequences observed in bacteria. These segments act as an immune system for bacteria against virus infections. CRISPR has found application in gene editing due to high flexibility and specific targeting. The technology has been widely implemented in stem cell research, gene therapy research, tissue and animal disease models, and various others. These advances in genetic research have witnessed rapid market acceptance as large number of research institutes and government agencies invested heavily in advanced research including stem cell research, gene therapy research, and various other research activities aimed at development of novel therapeutic. Hence introduction of new technologies assisting in these research activities receives swift acceptance in the research community. Wide spread awareness about the technology and its application has driven the CRISPR market globally. Simplicity of the technology and ease of use are other factors driving the CRISPR market growth and the growth for CRISPR services. Genomic engineering in cell lines is a multipurpose tool for researching biopharmaceutical research, designing diseases models, gene function, drug discovery among several other applications.Rapid growth in biotechnology and advances in genetic engineering has accelerated life science research. Increasing understanding of genetic makeup of various life forms and their role has provided research across the globe with solid grounds to discover novel therapeutic and commercially viable alternatives. With current alarming rise in incidence of chronic diseases such as cancer, diabetes, and various other cardiovascular and neurological disorders, there is an urgent need of long term treatment substitute. Advances in genetic research such as the CRISPR technology would help in achieving these milestones earlier.Request Free Report Sample@ www.sa-brc.com/Global-CRISPR-Products-and-Services-Market-Assessment--Forecast-2017-2021/up100Although low cost, and ease of use are beneficial for genetic research, scientists also believe that widespread and uncontrolled sale of such genetic engineering tools through e-commerce websites with increase the risk of bio-hacking. The CRISPR kits are available online at a price of less than US$ 200. This encourages uncontrolled and unauthorized use of biotechnology tools in modifying genetic sequences. Such research activities are likely to result in new disease causing agents that can affect large percentage of population globally. Various genetic researchers, advocacy groups and industry experts have demanded control of regulatory authorities for sale of CRISPR kits over e-commerce websites. Implementation of restraints on online sales would drastically affect the CRISPR market growth.Companies are seen investing heavily in providing cost efficient and CRISPR kits for more accurate results. In 2017, Synthego raised US$ 41 million to fortify its portfolio for CRISPR gene editing kits. Key players in the clustered regularly interspaced short palindromic repeats (CRISPR) market include ODIN, Thermo Fisher Scientific, Inc., OriGene Technologies, Inc., Cellecta, Inc., Takara Bio, System Biosciences, Inc., BioCat GmbH, Synthego Corporation, QIAGEN, Biolegio B.V. and various others.Request For TOC@ www.sa-brc.com/Global-CRISPR-Products--Services-Market-Assessment--Forecast-2017-2021/upcomingdetail100 Click here to view the list of recent Press Releases from SA-BRC
News Article | April 6, 2016
CRH-IRES-Cre mice were generated previously21. C57BL/6J wild-type mice, Emx1-IRES-Cre and Vglut2-IRES-Cre knock-in mice, and Rosa-floxstop-GFP reporter mice were purchased from the Jackson Laboratory. All procedures involving mice were approved by the Fred Hutchinson Cancer Research Center Institutional Animal Care and Use Committee. We used both males and females in all experiments, with similar numbers where possible. The same number of each sex was used in experiments shown in Fig. 1 and Extended Data Figs 2, 3, 5 and 6. No statistical methods were used to predetermine sample size. Animals were randomly chosen for experimental subjects. Animals were excluded from certain experiments. For nArc experiments shown in Fig. 2, animals were excluded if PRV+ cells were not found in all 5 OC and 3 VA areas analysed. For chemogenetic activation or silencing of the AmPir, animals were excluded if more than approximately 50% of mCherry+ cells were observed outside the AmPir. PRVs PRVB177 and PRVB316 were constructed using homologous recombination between targeting vectors and genomic DNA of PRV TK-BaBlu, a thymidine kinase (TK)-deleted PRV Bartha strain derivative with a LacZ insertion into the gG locus20. For targeting vectors, a flexstop-flanked sequence31 encoding a PRV TK fused at its C terminus to a haemagglutinin (HA) epitope tag (for PRVB177), or enhanced green fluorescent protein (eGFP) (for PRVB316), was first cloned with an inverse orientation into an eGFP-deleted pEGFP-N1 vector (Clontech). Next, NsiI fragments containing a CMV promoter, the flexstop-flanked coding sequence, and an SV40 polyadenylation signal were cloned between gG locus sequences matching those 5′ and 3′ to the lacZ sequence in PRV TK-BaBlu to give the final targeting vectors. These vectors were then linearized and co-transfected with PRV TK-BaBlu genomic DNA into HEK 293T cells (ATCC). Recombinant virus clones were selected and confirmed following methods described previously32. To propagate recombinant PRVs, PK15 cells (ATCC) were infected with the viruses using a multiplicity of infection (m.o.i.) = 0.1–0.01. After being infected, cells showed a prominent cytopathic effect (~2 days). They were harvested by scraping, and the cell material was frozen using liquid nitrogen and then quickly thawed in a 37 °C water bath. After three freeze–thaw cycles, cell debris was removed by centrifugation twice at 1,000g for 5 min and the supernatant was then used for experiments. The titre of viral stocks was determined using standard plaque assays on PK15 cells19, with titres expressed in plaque-forming units (p.f.u.). Lentivirus To generate LVF2TK, a flexstop-flanked sequence encoding TK–HA (see earlier) was cloned into the pLenti6.3 vector (Thermo Fisher). LVF2TK was produced using the ViraPower HiPerform Lentiviral Expression System (Thermo Fisher) according to the manufacturer’s instructions. Virus was concentrated using ultracentrifugation as described previously33. Viral titre was measured using the UltraRapid Lentiviral Titer Kit (System Biosciences) and titres were described in infectious units (i.f.u). AAVs Serotype 8 AAVs with Cre-recombinase-dependent flexstop cassettes that permit expression of mCherry-fused hM3Dq or mCherry-fused hM4Di under the control of the human synapsin promoter (AAV-DIO-hM3Dq-mCherry or AAV-DIO-hM4Di-mCherry) were purchased from the Vector Core at the University of North Carolina at Chapel Hill (the UNC vector core). Amount used is described in virus particles (v.p.). Mice aged 2–6 months were used for injection. Viruses were injected into the brain using a Stereotaxic Alignment System (David Kopf Instruments) with an inhalation anaesthesia of 2.5% isoflurane. Virus suspensions (PRVs: 1–1.5 × 106 p.f.u. (1 μl); LVF2TK: 1–1.5 × 106 i.f.u (1 μl); AAVs: 1–3 × 109 v.p. (200–330 nl)) were loaded into a 1-μl syringe, and injected at 100 nl per minute. The needle was inserted to the target locations based on a stereotaxic atlas30. After recovery, animals were singly housed with regular 12 h dark/light cycles, and food and water were provided ad libitum. Mice were exposed to a predator odour or distilled water within a modified polycarbonate vacuum-desiccator, as described previously34. A single mouse was placed on a platform in a chamber of 15 cm diameter with input and output ports, exposed to charcoal-filtered air for 16 h, and then to filtered air bubbled through 12 mM 2,5-dihydro-2,4,5-trimethylthiazoline (TMT) (Contech) diluted in water, water alone, bobcat urine (Maine outdoor solutions or Murray’s lures and trapping), or rabbit urine (Kishel’s quality animal scents and lures). For detection of nArc in PRV-infected cells, CRH-Cre mice injected with PRVB177 4 days earlier were exposed to odours for 5 min. For analysis of c-Fos expression in CRH neurons, CRH-Cre mice crossed with Rosa-floxstop-GFP mice were exposed to odours for 10 min and then to clean air for 50 min. All odour exposures were performed between 9:00 and 11:00 a.m. After mice were killed by cervical dislocation and decapitation, trunk blood was collected directly into blood collection tubes (Becton Dickinson) containing 50 μl aprotinin (Phoenix Pharmaceuticals). Plasma was obtained by centrifugation at 1,600g for 15 min at 4 °C, and stored at −80 °C. Plasma ACTH concentrations were measured using the ACTH ELISA kit (MD Biosciences), according to the manufacturer’s instructions, with the following modifications: (1) 100 μl of the controls or blood plasma combined with 100 μl of PBS (pH 7.4) was used in the place of 200 μl plasma; and (2) the results were assessed with the QuantaRed Enhanced Chemifluorescent HRP Substrate (Thermo Fisher). Plasma corticosterone concentrations were measured using the corticosterone ELISA kit (Abcam), according to the manufacturer’s instructions, with the following modifications: (1) plasma was diluted 25 times instead of 100 times with buffer M; and (2) the results were assessed with the QuantaRed Enhanced Chemifluorescent HRP Substrate. Fluorescence was measured with a CytoFluor4000 plate reader (Applied Biosystems). Animals were perfused transcardially with 4% paraformaldehyde (PFA). Their brains were then soaked in 4% PFA for 4 h, in 30% sucrose for 48 h, and then frozen in OCT (Sakura) and cut into 14–20-μm coronal sections using a cryostat. Brain sections were washed twice with PBS, permeabilized with 0.5% Triton X-100 in PBS for 5 min, washed twice with PBS, blocked with TNB (Perkin Elmer) for 1 h at room temperature, and then incubated with primary antibodies diluted in TNB at 4 °C overnight. Sections were then washed three times with TNT (0.1 M Tris pH 7.5, 150 mM NaCl, 0.05% Tween), incubated with the appropriate secondary antibodies and 0.5 μg ml−1 4′,6-diamidino-2-phenylindole (DAPI; Sigma) for 1 h at room temperature, and washed three times with TNT. Slides were coverslipped with Fluoromount-G (Southern Biotech). The following antibodies were used: (1) biotinylated mouse anti-HA (BioLegend, #901505, 1:300) followed by Alexa488-Streptavidin (Thermo Fisher, #S32354, 1:1,000) for polysynaptic PRV tracing; (2) goat anti-GFP (Rockland, #600-101-215, 1:1,000) and biotinylated anti-HA (BioLegend, 1:300) followed by Alexa488 donkey anti-goat IgG (Thermo Fisher, #A11055, 1:1,000) and Alexa555-Streptavidin (Thermo Fisher, #S32355, 1:1,000) for monosynaptic PRV tracing; (3) rabbit anti-GFP (Thermo Fisher, #A-11122, 1:500) and goat anti-c-Fos (Santa Cruz, #sc-52G, 1:300) followed by Alexa488 donkey anti-rabbit IgG (Thermo Fisher, #A21206, 1:1,000) and Alexa555 donkey anti-goat IgG (Thermo Fisher, #A21432, 1:1,000) for analysis of c-Fos expression in CRH neurons. In situ hybridization was performed essentially as described previously11, 35, with some experiments using additional steps for dual staining. Coding region fragments of Arc, Vglut1, Vglut2, Gad1, Gad2 and Crh, and the first intron sequence of c-Fos mRNA (for nuclear c-Fos staining) were isolated from mouse brain cDNA or mouse genomic cDNA using PCR, and cloned into the pCR4 Topo vector (Thermo Fisher). Digoxigenin (DIG)- or fluorescein (FLU)-labelled cRNA probes (riboprobes) were prepared using the DIG or FLU RNA Labelling Mix (Roche). Brains were frozen in OCT, and 16–20-μm coronal cryostat sections were hybridized to DIG- and/or FLU-labelled cRNA probes at 56 °C for 13–16 h. nArc mRNA staining After hybridization, sections were washed twice in 0.2 × SSC at 63 °C for 30 min, incubated with peroxidase (POD)-conjugated anti-DIG antibodies (Roche, #11207733910, 1:2,000) at 37 °C for 2 h, and then treated using the TSA-plus FLU kit (Perkin Elmer). Sections were then coverslipped with Fluoromount-G with DAPI (Southern Biotech). Co-staining for nArc, Vglut1/2 or Gad1/2 mRNA and HA (PRVB177) After hybridization, sections were washed twice in 0.2× SSC at 63 °C for 30 min, incubated with POD-conjugated anti-DIG antibodies (Roche, #11207733910, 1:2,000) and biotinylated anti-HA antibodies (BioLegend, #901505, 1:300) at 37 °C for 2 h, and then treated using the TSA-plus Cy3 kit (Perkin Elmer). Sections were then incubated with 0.5 μg ml−1 DAPI and Alexa488-Streptavidin (Thermo Fisher, 1:1,000) at room temperature for 1 h, and were coverslipped with Fluoromount-G. Co-staining with Crh and nuclear c-Fos riboprobes After hybridization, sections were washed twice in 0.2× SSC at 63 °C for 30 min, blocked with Streptavidin/Biotin Blocking kit (Vector Laboratories), incubated with POD-conjugated anti-FLU antibodies (Roche, #11426346910, 1:300) and alkaline phosphatase (AP)-conjugated anti-DIG antibodies (Roche, #11093274910, 1:300) at room temperature for 2 h, and then treated using the TSA-plus Biotin kit (Perkin Elmer). Sections were next incubated with 0.5 μg ml−1 DAPI and Alexa488-Streptavidin (Thermo Fisher, 1:1,000) at room temperature for 30 min, incubated with HNPP Fluorescent Detection Set (Roche) at room temperature for 1 h, and then coverslipped with Fluoromount-G. Activation AAV-DIO-hM3Dq-mCherry was injected into the pPir, AmPir or MEA of Emx1-IRES-Cre or Vglut2-IRES-Cre mice by stereotaxic injection (see earlier). At ≥2 weeks after injection, mice were intraperitoneally injected with clozapine-N-oxide (CNO; Sigma) (5.0 mg kg−1 body weight) or control saline. Twenty minutes later, trunk blood and brain were collected and the blood was used for plasma ACTH or corticosterone assays (see earlier). Brains were fixed with 4% PFA for 4 h, soaked in 30% sucrose for 24 h, frozen in OCT, and cut into 20-μm coronal sections using a cryostat. To analyse expression of hM3Dq-mCherry, brain sections were immunostained with rabbit anti-RFP (Rockland, #600-410-379, 1:500) followed by Alexa555 donkey anti-rabbit IgG (Thermo Fisher, #A31572, 1:1,000) antibodies (see earlier). Silencing AAV-DIO-hM4Di-mCherry was injected into the AmPir of Emx1-IRES-Cre mice by stereotaxic injection (see earlier). At ≥2 weeks after injection, mice were intraperitoneally injected with CNO (5.0 mg kg−1 body weight) or control saline. Forty minutes later, mice were exposed to bobcat urine or TMT for 10 min (see earlier). Animal behaviour during exposure was recorded with a Canon PowerShot ELPH300HS camera placed above the chamber, and the duration that mice stayed immobile during the 10-min odour exposure were scored by using Ethovision XT11 software (Noldus Information Technology). Trunk blood and brain were then collected. Blood was used for plasma ACTH or corticosterone assays (see earlier). Brains were treated and immunostained with rabbit-anti-RFP antibodies (see earlier). Images were collected using an AxioCam camera and AxioImager.Z1 microscope with apotome device (Zeiss), or TissueFAXS system (Tissuegnostics). Brain structures were identified microscopically and in digital photos using a mouse brain atlas30. To analyse the locations and number of PRV-infected cells in brain areas, every fifth section was analysed, and numbers of PRV+ cells were multiplied by five to acquire approximate total number of cells per animal. Brain areas were judged to contain upstream neurons if they contained ≥10 labelled neurons in ≥50% of animals. To analyse the location and number of virus-infected and nArc-expressing cells, three of five sections were analysed. The number of PRV+ cells with or without nArc mRNA was blindly counted. The percentages of PRV+ cells with only nArc signals among all PRV+ cells lacking cytoplasmic Arc were calculated. To analyse the activation of CRH neurons in chemogenetic experiments, the numbers of Crh+ neurons labelled for nc-Fos mRNA were blindly counted. To analyse the distribution of virus-infected cells, all sections through the regions of interest were analysed. The section images were aligned from anterior to posterior, and the numbers of virus-infected cells were counted. The size of each brain was adjusted linearly so that each posterior piriform cortex had the same length (2.8 mm). All data are shown as the mean ± s.e.m. Data were tested with the Shapiro–Wilk test for normality. For data with a normal distribution, the unpaired t-test or one-way ANOVA with post-hoc Dunnett’s test was used to compare two groups or more than two groups, respectively, to analyse statistical significance. For t-tests, equality of variances was analysed with the F-test and Welch’s correction was employed when variances of populations were significantly different. For data without a normal distribution, the Mann–Whitney U-test or the Kruskal–Wallis test with post-hoc Dunn’s test was used to compare two groups or more than two groups, respectively. All tests were two-sided. Detailed information on the numbers of animals used, statistical analyses, and effect sizes is provided in Supplementary Table 1. Abbreviations used for brain areas are according to Franklin and Paxinos30, with a few minor modifications. Non-olfactory areas AA, anterior amygdaloid area; AcbSh, accumbens nucleus, shell; AH, anterior hypothalamic area; AHi, amygdalohippocampal area; AI, agranular insular cortex; ARC, arcuate hypothalamic nucleus; AVPe, anteroventral periventricular nucleus; BLA, basolateral amygdala, anterior part; BLP, basolateral amygdaloid nucleus, posterior part; BMA, basomedial amygdala, anterior part; BMP, basomedial amygdaloid nucleus, posterior part; BNSTa, bed nucleus of the stria terminalis, anterior part; BNSTp, bed nucleus of the stria terminalis, posterior part; CA1, field CA1 of the hippocampus; CA3, field CA3 of the hippocampus; CEA, central amygdala; CEnt, caudomedial entothinal cortex; CI, caudal interstitial nucleus of the medial longitudinal; CVL, caudoventrolateral reticular nucleus; DEn, dorsal endopiriform claustrum; DI, dysgranular insular cortex; DMH, dorsomedial hypothalamic nucleus; DP, dorsal peduncular cortex; DRN, dorsal raphe nucleus; Ect, ectorhinal cortex; 7N, facial nucleu; Gi, gigantocellular reticular nucleus; HDB, nucleus of the horizontal limb of the diagonal band; I, intercalated nuclei of the amygdala; IPAC, interstitial nucleus of the posterior limb of the ant; IPC, interpeduncular nucleus, caudal subnucleus; IPR, interpeduncular nucleus, rostral subnucleus; IRt, intermediate reticular nucleus; La, lateral amygdala; LA, lateroanterior hypothalamic nucleus; LC, locus coeruleus; LDTg, laterodorsal tegmental nucleus; LH, lateral hypothalamic area; LPAG, lateral periaqueductal grey; LPGi, lateral paragigantocellular nucleus; LPO, lateral preoptic area; LRt, lateral reticular nucleus; LS, lateral septal nucleus; MCLH, magnocellular nucleus of the lateral hypothalamus; Md, medullary reticular nucleus; MEnt, medial entorhinal cortex; MnPO, median preoptic nucleus; MnR, median raphe nucleus; MPA, medial preoptic area; MPO, medial preoptic nucleus; MTu, medial tuberal nucleus; NLL, nucleus of the lateral lemniscus; NTS, nucleus of the solitary tract; p1PAG, p1 periaqueductal grey; PaS, parasubiculum; PBN, parabrachial nucleus; PCRt, parvicellular reticular nucleus; Pe, periventricular nucleus of the hypothalamus; PF, parafascicular thalamic nucleus; PH, posterior hypothalamic nucleus; PLH, peduncular part of lateral hypothalamus; PMD, premammillary nucleus, dorsal part; PMV, premammillary nucleus, ventral part; PO, paraolivary nucleus; Pr, prepositus nucleus; PRh, perirhinal cortex; PSTh, parasubthalamic nucleus; PVN, paraventricular nucleus of the hypothalamus; RCh, retrochiasmatic area; RM, retromammillary nucleus; SCh, suprachiasmatic nucleus; SHy, septohippocampal nucleus; SNC, substantia nigra; SO, supraoptic nucleus; StHy, striohypothalamic nucleus; STIA, bed nucleus of the stria terminalis, intraamygdaloid; STr, subiculum, transition area; SubC, subcoeruleus nucleus; VDB, vertical limb of the diagonal band; VLPO, ventrolateral preoptic nucleus; VMH, ventromedial hypothalamic nucleus; VP, ventral pallidum; VS, ventral subiculum; ZI, zona incerta.
News Article | January 27, 2016
C57BL/6J mice (CD45.2) and B6.SJL-PtprcaPep3b/BoyJ (CD45.1) were purchased from The Jackson Laboratory (Bar Harbour, ME). Prdm16Gt(OST67423)Lex knockout mice31 were obtained from Lexicon Genetics. Conditional Mito-Dendra2 transgenic (Pham) mice32 (B6;129S-Gt(ROSA)26Sortm1(CAG-COX8A/Dendra2)Dcc/J) and E2A-Cre mice33 (B6.FVB-Tg(EIIa-cre)C5379Lmgd/J) were purchased from Jackson Laboratory. Pham mice contain a mitochondrially targeted Dendra preceded by a stoplox sequence in the Rosa locus. These mice were crossed with E2A-Cre mice to effect ubiquitous induction of the MitoDendra2 reporter. Conditional Mfn2 knockout mice34 (B6/129SF1Mfn2tm3Dcc/Mmucd) were obtained from MMRRC and crossed to Vav-Cre transgenic mice35 (B6.Cg-Tg(Vav1-Cre)A2Kio/J) to obtain a homozygous floxed allele Mfn2 allele which generated a B6.Cg-Tg(Vav1-Cre)A2Kio/J;B6/126SF1 Mfn2tm3Dcc/Mmucd mixed mouse strain. All mouse strains were rederived by in vitro fertilization at the Jackson Laboratory. Animals were housed in a specific pathogen-free facility. Experiments and animal care were performed in accordance with the Columbia University Institutional Animal Care and Use Committee. All mice were used at age 8–12 weeks, except in experiments that involved fetal liver cells, when E14.4 embryos were used. Both sexes were used for experiments. Results were analysed in non-blinded fashion. In all experiments, randomly chosen wild type and littermates were used. MEFs were established from approximately 14.5 days post coitum embryos as previously described36 from Prdm16+/− breeder pairs. Briefly, dissected embryo trunks were minced into 1–2 mm fragments, resuspended in 3 ml 0.25% trypsin/EDTA (Gibco, Carlsbad, CA) and passed 20–30 times through a 16 gauge needle. Cell suspensions were incubated at 37 °C for 1 h with frequent agitation. Erythrocytes were lysed with ACK buffer, washed and cells were plated for 3 h in 10% FBS/DMEM. Cells remaining in suspension were aspirated and adherent cells were cultured with fresh media. MEFs were passaged 1:3 every 3 days and cells between passage 2 and 5 were used for all experiments. 293 cells and NIH-3T3 cells were purchased from ATCC (Manassas, VA) and sub-cultured in 10% FBS/DMEM or 10% calf serum/DMEM, respectively. WT and Mfn2−/− MEFs were a kind gift from E. Schon (Columbia University). All lines are tested yearly for mycoplasma contamination and found negative. Prdm16 constructs were generated by subcloning the murine full length (flPrdm16) or truncated (sPrdm16) cDNA into the XhoI/EcoRI sites of the pMSCV-IRES-GFP retroviral expression plasmid. The Mito-dsRed construct was purchased from Addgene (Cambridge, MA) (plasmid 11151). Mfn2 constructs were generated by subcloning the murine Mfn2 cDNA into the EcoRI/BamHI sites of the pLVX-EF1α-IRES-GFP or pLVX-EF1α-IRES-mCherry lentiviral expression plasmid (Clontech). The pGreenFire-Nfat and pGreenFire-CMV gene reporter constructs were purchased from System Biosciences (San Jose, CA) and contained three canonical Nfat response elements (5′- -3′) driving the expression of copGFP and luciferase reporters. The DNDrp1-pcDNA3.1 construct was purchased from Addgene (#45161) and subcloned using the BamHI/EcoRI restriction sites into the pLVX-IRES-GFP vector. Lentiviral 2nd generation packaging construct ΔR8.2 (8455) and pDM2.6 (12259) were purchased from Addgene. The −950/+22 murine MFN2 promoter was constructed by PCR amplification of the RP23-458J18 BAC clone (CHORI, Oakland, CA) and subcloned into the pGL4 luciferase reporter vector (Promega, Madison, WI). All cloning was carried out using KOD hot-start polymerase (Novagen, Billerica, MA) and subcloned for screening and sequencing into the pCR2.1 shuttle vector (Invitrogen, Carlsbad, CA). For peripheral blood analyses, erythrocytes were lysed twice with ACK lysis buffer and nucleated cells were stained with antibody cocktail (Supplementary Table 1) in FACS buffer for 15 min on ice, washed and analysed on a BD FACSCantoII flow cytometer (Becton Dickinson, Mountain View, CA). For bone marrow analyses, cells were isolated using the crushing method and erythrocytes were lysed with ACK lysis buffer followed by 40 μm filtration. bone marrow cells were stained with antibody cocktail in FACS buffer for 30 min on ice, washed and analysed on a BD LSRII flow cytometer (Becton Dickinson, Mountain View, CA). Dead cells were excluded from analyses by gating out 7AAD-positive cells. To isolate purified haematopoietic populations, bone marrow cells were isolated, stained and sorted using a BD Influx cell sorter (Becton Dickinson, Mountain View, CA) into complete media. Data were analysed using FlowJo9.6 (TreeStar Inc., Ashland, OR). Mfn2fl/fl-Vav-Cre fetal liver cells, bone marrow cells or purified LT-HSCs (Lin−cKit+Sca1+CD48−Flt3−CD150+) were transplanted into lethally irradiated (two doses of 478 cGy over 3 h using a Rad Source RS-2000 X-ray irradiator (Brentwood, TN)) recipients together with 2 × 105 competitor cells. As Mfn2fl/fl-Vav-Cre mice were not fully backcrossed onto the C57BL/6 background, recipient mice and competitor bone marrow cells were from the B6.Cg-Tg(Vav1-Cre)A2Kio/J;B6/126SF1 Mfn2tm3Dcc/Mmucd mixed background mouse strain crossed to B6.SJL-Ptprca Pep3b/BoyJ (CD45.1) to generate a CD45.1+CD45.2+ mixed background mouse. Competitor cells were T-cell depleted using MACS beads. For all competitive transplantation experiments, at least two independent transplants, each with at least 4 recipients per condition of genotype were performed, and result of all recipients pooled for statistical analysis. Power calculation was based on results of the first experiment. In limiting dilution assays, cohorts of recipients received 20 or 50 HSCs together with 2 × 105 competitor cells, allowing calculation of HSC frequency based on the number of non-repopulated mice (<1% donor contribution) using Poisson statistics 15 weeks after reconstitution. For Mfn2 KO single cell transplantation, LT-HSCs were sorted directly into complete media (StemPro34, 100 ng ml−1 SCF, 100 ng ml−1 TPO, 50 ng ml−1 IL-6) and single cells were visually confirmed. Positive single cell wells were combined with 2 × 105 CD45.1 competitor bone marrow cells and transplanted into lethally irradiated CD45.1 recipient mice. Recipients showing ≥ 0.1% CD45.2 donor contribution were considered positive and GM/(B+T) ratios were calculated as previously described for characterizing heterogeneous HSC phenotypes37. In transplantations using WT or Prdm16−/− HSCs (Lin−cKit+Sca1+CD48−Flt3−CD150+) B6.CD45.2 cells were mixed with 2 × 105 freshly isolated B6.CD45.1 bone marrow cells and injected via tail vein into lethally irradiated (two doses of 478 cGy over 3 h using a Rad Source RS-2000 X-ray irradiator (Brentwood, TN)) B6.CD45.1+CD45.2+ F1 hybrid recipients. After 8 to 15 weeks, peripheral blood (PB) and bone marrow were analysed. Lentiviral particles were produced by seeding 293 cells at 7 × 105 per cm2, or PlatE cells (Cell Biolabs, San Diego, CA), in Ultra Culture serum-free media (Lonza, Basel, Switzerland) overnight followed by transfection of each packaging and expression construct (1:1:1) using Trans-It 293 (Mirus, Madison, WI) for 2 h. Media were pooled after 36–48 h, clarified and concentrated by ultracentrifugation (100,000g), resuspended in StemPro-34 media and stored at −80 °C. Virus titre was calculated from transduction of NIH-3T3 fibroblasts serial dilutions of the viral preparation. Sorted LT-HSCs were transduced with ≥ 150 MOI lentivirus particles in the presence of 6 μg ml−1 polybrene (Sigma) and spun at 900g for 20 min at 20 °C. Supernatant was aspirated and replaced with complete media and cultured overnight. Transduction efficiency of cells was confirmed after 24 h. To assess proviral copy number 15 weeks post-transplantation in vivo, splenocytes were harvested and sorted into donor (CD45.2) or competitor (CD45.1) populations and gDNA was isolated as previously described38. Amplification of the proviral WPRE region was achieved using SYBR Green qPCR assay using the primer pair WPREFor: 5′- -3′ and WPRERev: 5′- -3′. Quantification of proviral copies was derived from the linear regression of serial dilutions of viral vector and normalized to input cell number. Sorted or cultured cell populations (2–5 × 103 cells) were lysed in TRIzol LS reagent (Invitrogen, Carlsbad, CA) and RNA was isolated according to manufacturer’s instructions. cDNA was synthesized using Superscript III Reverse Transcriptase (Invitrogen) and target CT values were determined using inventoried TaqMan probes (Applied Biosystems, Carlsbad, CA, see Supplementary Table 2) spanning exon/exon boundaries and detected using a Viia7 Real Time PCR System (Applied Biosystems). Relative quantification was calculated using the ΔΔC method. To estimate relative copy number of Mfn1 and Mfn2 transcripts (Fig. 4a), copy numbers were derived from the linear regression of serial dilutions of respective cDNA plasmids and normalized to GAPDH-VIC values. To estimate relative copy number of flPrdm16 transcripts (Fig. 4d), a probe was designed to span the SET methyltransferase domain of Prdm16 (exon2/3 junction) and copy number was derived from the linear regression of serial dilutions of respective cDNA plasmids. Another probe (exon 14/15 junction) was used to quantify total Prdm16 copy numbers derived from the linear regression of serial dilutions of respective cDNA plasmids. The values derived from total Prdm16 probe was subtracted from flPrdm16-specific probe to determine sPrdm16 transcript quantity. All values were normalized to relative multiplexed GAPDH-VIC values. Culture of sorted LT-HSCs was carried out using StemPro34 media (Invitrogen) supplemented with 10 mM HEPES and 50 ng ml−1 of recombinant murine SCF, TPO, IL-6 (Peptrotech, Rocky Hill, NJ) and cultured in 5% O at 37 °C. In some experiments, LT-HSCs were cultured in the presence of 500 ng ml−1 VIVIT (Millipore, Billerica, MA) or 30 μM mDivi1 (MolPort, Riga, Latvia). To demonstrate a mitochondrial fusion activity, cell fusion experiments were performed using MEFs as previously described37. Briefly, BacMam baculovirus constructs (Invitrogen) expressing the signalling peptide from cytochrome c fused to either GFP or RFP were transduced separately into MEF cells. Sorted GFP+ and RFP+ MEFs were co-cultured for 24 h and plasma membranes were fused using PEG-1500 (Roche, Basel, Switzerland. Fused cells were cultured in DMEM containing cyclohexamide (Sigma, St. Louis, MO) for 4 h and analysed for colocalization of mitochondrial labels. Early passage Prdm16−/− MEFs were transduced with 10 MOI retrovirus for 72 h and fixed with 4% paraformaldehyde for 10 min. Protein lysates were isolated and chromatin immunoprecipitation was carried out using the ChIP-IT Express Enzyme kit (Active Motif, Carlsbad, CA). Antibodies used for ChIP include anti-Flag and anti-TF2D. Primer probes were designed to span regions of the Mfn2 promoter previously shown to regulate Mfn2 transcriptional activity (see Supplementary Table 3)39. Quantification of precipitated Mfn2 promoter regions were derived from the linear regression of serial dilutions of bone marrow genomic DNA, normalized to input DNA concentration and quantifiable IgG detection was subtracted from sample values. Bone marrow was freshly isolated and lineage depleted with the MACS Lineage Depletion Kit (Miltenyi Biotech, San Diego, CA). Cells were cultured for 30 min in complete medium supplemented with 1 μM Indo-1 prepared as stock supplemented with Pluronic-F127 and incubated at 37 °C for 30 min. Cells were washed and stained for surface markers for 15 min, washed and allowed to rest in for 15 min PBS in PBS with Ca2+. FACS tubes were run at 37 °C in the sample port of the LSRII flow cytometer equipped with a 355 nm excitation laser. Events were collected for 40 s before incubation with 25 μM ATP or 1 μM SDF1 to induce calcium transients. The average ratio, R, of bound/free Indo-1 (405 nm/485 nm emission) before simulation was used to determine baseline values. Identical samples were equilibrated in 10 mM EGTA PBS without Ca2+ to determine R or stimulated with 1 μM ionomycin to determine R . The Indo-1 dissociation constant (K ) was assumed to be 237 nM at 37 °C based on previous studies40. The following equation was then used to relate Indo-1 intensity ratios to [Ca2+] levels; Sorted or cultured haematopoietic populations (2–5 × 103 cells) were collected in complete media and plated on onto MicroWell 96-well glass-bottom plates (Thermo, Waltham, MA) coated with 1ug ml−1 poly-d-lysine. Cells were allowed to adhere for 10 min and fixed with 4% PFA for 15 min. Cells were then permeabilized with 0.1% TritonX-100/PBS for 5 min and blocked with 2% BSA/PBS for 1 h at 4 °C. Cells were incubated with anti-Nfat1 (1:100), anti-Mfn2 (1:200), anti-tubulin (1:200), anti CD150-APC (1:100) or anti-Flag (1:250) (see Supplementary Table 1) overnight, washed and incubated with AlexaFluor secondary antibodies (Invitrogen) for 1 h. Cell nuclei were counterstained with DAPI and mounted with fluorescent mounting media (Vector Labs, Burlingame, CA). Confocal images were acquired with a Zeiss LSM 700 confocal microscope or a Leica DMI 6000B and images were deconvoluted and processed with Leica AF6000 software package. NIH-3T3, WT or Mfn2−/− MEF cells were plated at 2 × 104 cells per cm2 in triplicate overnight and transfected with 500 ng of pGF-Nfat, pGF-CMV or −950/+22 Mfn2-pGL4 reporter construct, 500 ng of cDNA plasmids as indicated and 500 ng of either pSV-βGal or pLVX-IRES-mCherry plasmids with Lipofectamine 3000 according to manufacturer’s instructions for 24 or 48 h. Cells were lysed in reporter lysis buffer (Promega, Madison, WI) and analysed for luciferase activity using BrightGlo luciferase (Promega) and detected on a Synergy H2 plate reader (BioTek, Winooski, VT). To visualize βGal activity, cell lysate was incubated in Buffer Z (1mg ml−1 ONPG, 0.1 M phosphate, pH 7.5, 10 mM KCl, 1 mM βME, 1 mM MgSO ) at 37 °C for 1 h. Absorbance values were measured at 405 nm and used to normalize for transfection efficiency. In WT and Prdm16−/− MEFs, gene reporter luciferase values were normalized to mCherry excitation values. For total cell lysate experiments, MEF cultures were lysed in RIPA buffer, 50 mM Tris pH 7.5, 137 mM NaCl, 0.1% SDS, 0.5% deoxycholate and protease inhibitors (Roche). For subcellular fractionation studies, cells were scraped, washed in PBS. Cell pellets were lysed in 5× packed cell volume (pcv) Buffer A for 10 min on ice and vortexed for 15 s in the presence of 1/10 volume 3% NP-40. Plasma membrane lysis was verified by trypan blue staining. Lysate was spun at 15,000g for 10 min at 4 °C and the cytoplasmic fraction was saved. The remaining nuclear pellet was resuspended in 2.5× pc Buffer C and incubated at 4 °C for 1 h with rotation and spun at 15,000g for 10 min. The nuclear fraction was diluted with 2.5× volume of Nuclear Diluent Buffer and stored at −80 °C. To achieve even fractionation loading, equivalent percentages of nuclear and cytoplasmic fractions were loaded on each gel. All protein samples were denatured in 4× sample buffer at 95 °C and loaded onto 4–12% Bis-Tris SDS–PAGE gradient gels (Invitrogen). Gels were transferred onto 0.22 μm nitrocellulose membrane and stained with Ruby Red (Molecular Probes, Carlsbad, CA) to confirm transfer. Membranes were blocked with 3% non-fat milk or BSA in 0.1%Tween-20/TBS and incubated with anti-Mfn2 (1:200), anti-βGal (1:1,000), anti-Nfat1 (1:250), anti-tubulin (1:1,000), anti-lamin A/C (1:500) and anti-β-actin (1:5,000) overnight (see Supplementary Table 1). Membranes were washed, incubated with HRPO-conjugated secondary antibodies and exposed to X-ray film (Denville) after incubation with Super Signal West Fempto ECL reagent (Pierce). For mitochondrial length measurements, confocal or deconvoluted z-stacks were collected and projected as a z-project in ImageJ (NIH, Bethesda, MD). Individual mitochondria were manually traced, binned into length categories and expressed as percent of cellular mitochondria. The mean ± s.e.m. number of mitochondria falling into each length category collected from ≥ 15 fields (30–50 cells) are expressed. For Nfat nuclear localization quantification, confocal or deconvoluted z-stacks were collected and a 1-μm section in the centre of the cell was projected as a z-project in ImageJ. Nuclear boundaries were constructed using DAPI staining. The ratio of staining within the nuclear boundary to total staining was expressed as percent of Nfat signal. The mean ± s.e.m. for ≥ 10 fields (20–40 cells) are expressed. For immunofluorescence intensity measurements, confocal or deconvoluted z-stacks were collected and projected as a z-project in ImageJ. Thresholds were set based on IgG-stained negative control cells and the integrated density value of each signal per cell was recorded. The mean ± s.e.m. for ≥ 15 fields (30–50 cells) are expressed. For statistical analysis between two groups, the unpaired Student’s t-test was used. When more than two groups were compared, one-way ANOVA was used. Results are expressed as mean ± s.e.m. The Bonferroni and Dunnett multiple comparison tests were used for post-hoc analysis to determine statistical significance between multiple groups. All statistics were calculated using Prism5 (GraphPad, La Jolla, CA) software. Differences among group means were considered significant when the probability value, P, was less than 0.05. Sample size (‘n’) always represents biological replicates. Cochran test was used for exclusion of outliers. No statistical methods were used to predetermine sample size. The experiments were not randomized, and the investigators were not blinded to allocation during experiments and outcome assessment.
News Article | November 4, 2015
All common chemicals were from Sigma. Pyrrolidinedithiocarbamic acid was from Santa Cruz Biotechnology. Exo-FBS exosome-depleted FBS was purchased from System Biosciences (SBI). PTEN (9188), pAkt(T308) (9275), pAkt(S473) (4060), Pan Akt (4691), and Bim (2933) antibodies were from Cell Signaling. CD9 (ab92726), Rab27a (ab55667), AMPK (ab3759), CCL2 (ab9899), MAP2 (ab11267), and pP70S6K (ab60948) antibodies were from Abcam. Tsg101 (14497-1-AP) and Rab27b (13412-1-AP) antibodies were from Proteintech. CD81 (104901) antibody was from BioLegend. E2F1 (NB600-210) and CCR2 (NBP1-48338) antibodies were from Novus. GFAP (Z0334) antibody was from DAKO. IBA1 antibody was from WAKO. Cre (969050) antibody was from Novagen. NF-κB p65 (SC-109) and CD63 (SC-15363) antibodies were from Santa Cruz. DMA (sc-202459) and CCR2 antagonist (sc-202525) were from Santa Cruz. MK2206 (S1078) was from Selleckchem. PDTC (P8765) was from Sigma-Aldrich. Human breast cancer cell lines (MDA-MB-231, HCC1954, BT474 and MDA-MB-435) and mouse cell lines (B16BL6 mouse melanoma and 4T1 mouse breast cancer) were purchased from ATCC and verified by the MD Anderson Cancer Center (MDACC) Cell Line Characterization Core Facility. All cell lines have been tested for mycoplasma contamination. Primary glia was isolated as described13. In brief, after homogenization of dissected brain from postnatal day (P)0–P2 neonatal mouse pups, all cells were seeded on poly-d-lysine coated flasks. After 7 days, flasks with primary culture were placed on an orbital shaker and shaken at 230 r.p.m. for 3 h. Warm DMEM 10:10:1 (10% of fetal bovine serum, 10% of horse serum, 1% penicillin/streptomycin) was added and flasks were shaken again at 260 r.p.m. overnight. After shaking, fresh trypsin was added into the flask and leftover cells were plated with warm DMEM 5:5:1 (5% of fetal bovine serum, 5% of horse serum, 1% penicillin/streptomycin) to establish primary astrocyte culture. More than 90% of isolated primary glial cells were GFAP+ astrocytes. Primary CAFs were isolated by digesting the mammary tumours from MMTV-neu transgenic mouse. 231-xenograft CAFs were isolated by digesting the mammary tumours from MDA-MB-231 xenograft. For the mixed co-culture experiments, tumour cells were mixed with an equal number of freshly isolated primary glia, CAFs or NIH3T3 fibroblast cells in six-well plate (1:3 ratio). Co-cultures were maintained for 2–5 days before magnetic-bead-based separation. For the trans-well co-culture experiments, tumour cells were seeded in the bottom well and freshly isolated primary glia, CAFs or NIH3T3 cells were seeded on the upper insert (1:3 ratio). Co-cultures were maintained for 2–5 days for the further experiments. Lentiviral-based packaging vectors (Addgene), pLKO.1 PTEN-targeting shRNAs and all siRNAs (Sigma), Human Cytokine Antibody Array 3 (Ray biotech), and lentiviral-based vector pTRIPZ-PTEN and pTRIPZ-CCL2 shRNAs (MDACC shRNA and ORFome Core, from Open Biosystems) were purchased. The human PTEN-targeting shRNA sequences in the lentiviral constructs were: 5′-CCGGAGGCGCTATGTGTATTATTATCTCGAGATAATAATACACATAGCGCCTTTTTT-3′ (targeting coding sequence); 5′-CCGGCCACAAATGAAGGGATATAAACTCGAGTTTATATCCCTTCATTTGTGGTTTTT-3′ (targeting 3′-UTR). The human PTEN-targeting siRNA sequences used were: 5′-GGUGUAAUGAUAUGUGCAU-3′ and 5′-GUUAAAGAAUCAUCUGGAU-3′. The human CCL2-targeting siRNA sequences used were: 5′-CAGCAAGUGUCCCAAAGAA-3′ and 5′-CCGAAGACUUGAACACUCA-3′. The mouse Rab27a-targeting siRNA sequences used were: 5′-CGAUUGAGAUGCUCCUGGA-3′ and 5′-GUCAUUUAGGGAUCCAAGA-3′. Mouse pLKO shRNA (shRab27a: TRCN0000381753; shRab27b: TRCN0000100429) were purchased from Sigma. For lentiviral production, lentiviral expression vector was co-transfected with the third-generation lentivirus packing vectors into 293T cells using Lipo293 DNA in vitro Transfection Reagent (SignaGen). Then, 48–72 h after transfection, cancer cell lines were stably infected with viral particles. Transient transfection with siRNA was performed using pepMute siRNA transfection reagent (SignaGen). For in vivo intracranial virus injection, lentivirus was collected from 15 cm plates 48 h after transfection of packaging vectors. After passing a 0.45 μm filter, all viruses were centrifuged at 25,000 r.p.m (111,000g) for 90 min at 4 °C. Viral pellet was suspended in PBS (~200-fold concentrated). The final virus titre (~1 × 109 UT ml−1) was confirmed by limiting dilution. Cell isolation was performed based on the magnetic bead-based cell sorting protocol according to manufacturer’s recommendation (Miltenyi Biotec Inc.). After preparation of a single-cell suspension, tumour cells (HCC1954 or BT474) were stained with primary EpCAM-FITC antibody (130-098-113) (50 μl per 107 total cells) and incubated for 30 min in the dark at 4 °C. After washing, the cell pellet was re-suspended and anti-FITC microbeads (50 μl per 107 total cells) were added before loading onto the magnetic column of a MACS separator. The column was washed twice and removed from the separator. The magnetically captured cells were flushed out immediately by firmly applying the plunger. The isolated and labelled cells were analysed on a Gallios flow cytometer (Beckman Coulter). For EpCAM-negative MDA-MB-231 tumour cells, FACS sorting (ARIAII, Becton Dickinson) was used to isolate green fluorescent protein (GFP)+ tumour cells from glia or CAFs. Isolation of primary glia was achieved by homogenization of dissected brain from P0–P2 mouse pups. After 7 days, trypsin was added and cells were collected. After centrifugation and re-suspension of cell pellet to a single-cell suspension, cells were incubated with CD11b+ microbeads (Miltenyl Biotec) (50 μl per 107 total cells) for 30 min at 4 °C. The cells were washed with buffer and CD11b+ cells were isolated by MACS Column. CD11b+ cells were analysed by flow cytometry and immunofluorescence staining. Western blotting was done as previously described. In brief, cells were lysed in lysis buffer (20 mM Tris, pH 7.0, 1% Triton X-100, 0.5% NP-40, 250 mM NaCl, 3 mM EDTA and protease inhibitor cocktail). Proteins were separated by SDS–PAGE and transferred onto a nitrocellulose membrane. After membranes were blocked with 5% milk for 30 min, they were probed with various primary antibodies overnight at 4 °C, followed by incubation with secondary antibodies for 1 h at room temperature, and visualized with enhanced chemiluminescence reagent (Thermo Scientific). In brief, total RNA was isolated using miRNeasy Mini Kit (Qiagen) and then reverse transcribed using reverse transcriptase kits (iScript cDNA synthesis Kit, Bio-rad). SYBR-based qRT–PCR was performed using pre-designed primers (Life Technologies). miRNA assay was conducted using Taqman miRNA assay kit (Life Technologies). For quantification of gene expression, real-time PCR was conducted using Kapa Probe Fast Universal qPCR, and SYBR Fast Universal qPCR Master Mix (Kapa Biosystems) on a StepOnePlus real-time PCR system (Applied Biosystems). The relative expression of mRNAs was quantified by 2−ΔΔCt with logarithm transformation. Primers used in qRT–PCR analyses are: mouse Ccl2: forward, 5′-GTTGGCTCAGCCAGATGCA-3′; reverse: 5′-AGCCTACTCATTGGGATCATCTTG-3′. Mouse Actb: forward: 5′-AGTGTGACGTTGACATCCGT3′; reverse: 5′-TGCTAGGAGCCAGAGCAGTA-3′. Mouse Pten: forward: 5′-AACTTGCAATCCTCAGTTTG-3′; reverse: 5′-CTACTTTGATATCACCACACAC-3′. Mouse Ccr2 primer: Cat: 4351372 ID: Mm04207877_m1 (Life technologies) Synthetic miRNAs were purchased from Sigma and labelled with Cy3 by Silencer siRNA labelling kit (Life Technologies). In brief, miRNAs were incubated with labelling reagent for 1 h at 37 °C in the dark, and then labelled miRNAs were precipitated by ethanol. Labelled miRNAs (100 pmoles) were transfected into astrocytes or CAFs in a 10-cm plate. After 48 h, astrocytes and CAFs containing Cy3-miRNAs were co-cultured with tumour cells (at 5:1 ratio). Genomic DNA was isolated by PreLink genomic DNA mini Kit (Invitrogen), bisulfite conversion was performed by EpiTect Bisulphite Kit and followed by EpiTect methylation-specific PCR (Qiagen). Primers for PTEN CpG island are 5′-TGTAAAACGACGGCCAGTTTGTTATTATTTTTAGGGTTGGGAA-3′ and 5′-CAGGAAACAGCTATGACCCTAAACCTACTTCTCCTCAACAACC-3′. Luciferase reporter assays were done as previously described27. The wild-type PTEN promoter driven pGL3-luciferase reporter was a gift from A. Yung. The pGL3-PTEN reporter and a control Renilla luciferase vector were co-transfected into tumour cells by Lipofectamine 2000 (Life Technologies). After 48 h, tumour cells were co-cultured with astrocytes or CAFs. Another 48 h later, luciferase activities were measured by Dual-Luciferase Report Assay Kit (Promega) on Luminometer 20/20 (Turner Biosystems). The PTEN 3′-UTRs with various miRNA binding-site mutations were generated by standard PCR-mediated mutagenesis method and inserted downstream of luciferase reporter gene in pGL3 vector. The activities of the luciferase reporter with the wild-type and mutated PTEN 3′-UTRs were assayed as described above. Astrocytes or CAFs were cultured for 48–72 h and exosomes were collected from their culture media after sequential ultracentrifugation as described previously. In brief, cells were collected, centrifuged at 300g for 10 min, and the supernatants were collected for centrifugation at 2,000g for 10 min, 10,000g for 30 min. The pellet was washed once with PBS and purified by centrifugation at 100,000g for 70 min. The final pellet containing exosomes was re-suspended in PBS and used for (1) transmission electron microscopy by fixing exosomes with 2% glutaraldehyde in 0.1 M phosophate buffer, pH 7.4; (2) measure of total exosome protein content using BCA Protein Assay normalized by equal number of primary astrocytes and CAF cells; (3) western blotting of exosome marker protein CD63, CD81 and Tsg101; and (4) qRT–PCR by extracting miRNAs with miRNeasy Mini Kit (Qiagen). Fixed samples were placed on 100-mesh carbon-coated, formvar-coated nickel grids treated with poly-l-lysine for about 30 min. After washing the samples on several drops of PBS, samples were incubated on drops of buffered 1% gluteraldehyde for 5 min, and then washed several times on drops of distilled water. Afterwards, samples were negatively stained on drops of millipore-filtered aqueous 4% uranyl acetate for 5 min. Stain was blotted dry from the grids with filter paper and samples were allowed to dry. Samples were then examined in a JEM 1010 transmission electron microscope (JEOL) at an accelerating voltage of 80 Kv. Digital images were obtained using the AMT Imaging System (Advanced Microscopy Techniques Corp.). For exosome detection, 100 μl exosomes isolated from 10-ml conditioned media of astrocytes or CAFs were incubated with 10 μl of aldehyde/sulfate latex beads (4 μm diameter, Life Technologies) for 15 min at 4 °C. After 15 min, PBS was added to make sample volume up to 400 μl, which was incubated overnight at 4 °C under gentle agitation. Exosome-coated beads were washed twice in FACS washing buffer (1% BSA and 0.1% NaN in PBS), and re-suspended in 400 μl FACS washing buffer, stained with 4 μg of phycoerythrin (PE)-conjugated anti-mouse CD63 antibody (BioLegend) or mouse IgG (Santa Cruz Biotechnology) for 3 h at 4 °C under gentle agitation and analysed on a FACS Canto II flow cytometer. Samples were gated on bead singlets based on FCS and SSC characteristics (4 μm diameter). For Annexin V apoptosis assay, after 24 h doxorubicin (2 μM) treatment, the cells were collected, labelled by APC-Annexin V antibody (Biolegend) and analysed on a FACS Canto II flow cytometer. CD11b+ and BV2 cells were stained with CCR2 antibody (Novus) at 4 °C overnight; they were then washed and stained with Alexa Fluor 488 anti-rabbit IgG (Life Technologies) at room temperature for 1 h. The cells were then analysed on a FACS Canto II flow cytometer. All animal experiments and terminal endpoints were carried out in accordance with approved protocols from the Institutional Animal Care and Use Committee of the MDACC. Animal numbers of each group were calculated by power analysis and animals are grouped randomly for each experiment. No blinding of experiment groups was conducted. MFP tumours were established by injection of 5 × 106 tumour cells in 100 μl of PBS:Matrigel mixture (1:1 ratio) orthotopically into the MFP of 8-week-old Swiss nude mice as done previously28. Brain metastasis tumours were established by ICA injection of tumour cells (250,000 cells in 0.1 ml HBSS for MDA-MB-231, HCC1954, MDA-MB-435, 4T1 and B16BL6, and 500,000 cells in 0.1 ml HBSS for BT474.m1 into the right common carotid artery as done previously29). Mice (6–8 weeks) were randomly grouped into designated groups. Female mice are used for breast cancer experiments, both female and male are used for melanoma experiments. Since the brain metastasis model does not result in visible tumour burdens in living animal, the endpoints of in vivo metastasis experiments are based on the presence of clinical signs of brain metastasis, including but not limited to, primary central nervous system disturbances, weight loss, and behavioural abnormalities. Animals are culled after showing the above signs or 1–2 weeks after surgery based on specific experimental designs. Brain metastasis lesions are enumerated as experimental readout. Brain metastases were counted as micromets and macromets. The definition of micromets and macromets are based on a comprehensive mouse and human comparison study previously published30. In brief, ten haematoxylin and eosin (H&E)-stained serial sagittal sections (300 μm per section) through the left hemisphere of the brain were analysed for the presence of metastatic lesions. We counted micrometastases (that is, those ≤ 50 μm in diameter) to a maximum of 300 micrometastases per section, and every large metastasis (that is, those > 50 μm in diameter) in each section. Brain-seeking cells from overt metastases and whole brains were dissected and disaggregated in DMEM/F-12 medium using Tenbroeck homogenizer briefly. Dissociated cell mixtures were plated on tissue culture dish. Two weeks later, tumours cells recovered from brain tissue were collected and expanded as brain-seeking sublines (Br.1). For the astrocyte miR-19 knockout mouse model, Mirc1tm1.1Tyj/J mice (Jax lab) (6–8 weeks) were intracranially injected with Ad5-GFAP-Cre virus (Iowa University, Gene Transfer Vector Core) 2 μl (MOI ~108 U μl−1) per point, total four points at the right hemisphere (n = 9). Control group (n = 7) was injected with the same dose Ad5-RSV-βGLuc (Ad-βGLuc) at the right hemisphere. All intracranial injections were performed by an implantable guide-screw system. One week after virus injection, mice were intracarotidly injected with 2 × 105 B16BL6 tumour cells. After two weeks, whole brains were dissected and fixed in 4% formaldehyde, and embedded in paraffin. Tumour formation, histological phenotypes of H&E-stained sections, and IHC staining were evaluated. Only parenchymal lesions, which are in close proximity of adenovirus injection, were included in our evaluation. Tumour size was calculated as (longest diameter) × (shortest diameter)2/2. For the intracranial tumour model, Mirc1tm1.1Tyj/J mice (Jax lab) (6–8 weeks) were intracranially injected as described above. Seven mice were used in the experiment. One week later, these mice were intracranially injected with 2.5 × 105 B16BL6 tumour cells at both sides where adenoviruses were injected. After another week, whole brains were dissected and fixed in 4% formaldehyde, and embedded in paraffin. Tumour formation and phenotype were analysed as above. For the Rab27a/b knockdown mouse model, seven C57BL6 mice (Jax lab) (6–8 weeks) were intracranially injected with concentrated lentivirus containing shRab27a and shRab27b (ratio 1:2) 2 μl per point, total three points at the right hemisphere; concentrated control lentivirus containing pLKO.1 scramble were injected at the left hemisphere. All intracranial injections were performed by an implantable guide-screw system. One week later, mice were intracranially injected with 5 × 104 B16BL6 tumour cells at both sides where they had been infected. After one week, whole brains were dissected and fixed in 4% formaldehyde, and embedded in paraffin. Tumour formation, histological phenotypes of H&E-stained sections, IHC staining were evaluated. When performing metastases size quantification, only parenchymal lesions that were in close proximity to the adenovirus injection sites were included in the analyses. Tumour size was calculated as (longest diameter) × (shortest diameter)2/2. For exosome rescue experiments, eight C57BL6 mice (Jax lab) (6–8 weeks) were intracranially injected with concentrated lentivirus containing shRab27a and shRab27b (ratio 1:2) 2 μl per point, total 3 points at both hemispheres. One week later, these mice were intracranially injected with 5 × 104 B16BL6 tumour cells with 10 μg exosome isolated from astrocyte media at the right sides where they had been injected with lentivirus; 5 × 104 B16BL6 tumour cells with vehicle were injected at the left sides where lentivirus had been injected. After another week, whole brains were dissected and fixed in 4% formaldehyde, and embedded in paraffin. Tumour formation and phenotype were analysed as above. For in vivo extravasation assay, equal numbers of cells labelled with GFP-control shRNA and RFP-PTEN shRNA (Open Biosystems) were mixed and ICA injected. After cardiac perfusion, brains were collected and sectioned through coronal plan on a vibrotome (Leica) into 50-μm slices. Fluorescent cells were then counted. For inducible PTEN expression in vivo, mice were given doxycycline (10 μg kg−1) every other day. To quantify brain metastasis incidence and tumour size, brains were excised for imaging and histological examination at the end of experiments. Ten serial sagittal sections every 300 μm throughout the brain were analysed by at least two pathologists who were blinded to animal groups in all above analyses. Reverse-phase protein array of PTEN-overexpressing cells was performed in the MDACC Functional Proteomics core facility. In brief, cellular proteins were denatured by 1% SDS, serial diluted and spotted on nitrocellulose-coated slides. Each slide was probed with a validated primary antibody plus a biotin-conjugated secondary antibody. The signal obtained was amplified using a Dako Cytomation-catalysed system and visualized by DAB colorimetric reaction. The slides were analysed using customized Microvigene software (VigeneTech Inc.). Each dilution curve was fitted with a logistic model (‘Super curve fitting’ developed at the MDACC) and normalized by median polish. Differential intensity of normalized log values of each antibody between RFP (control) and PTEN-overexpressed cells were compared in GenePattern (http://genepattern.broadinstitute.org). Antibodies with differential expression (P < 0.2) were selected for clustering and heat-map analysis. The data clustering was performed using GenePattern. Two studies in separate cohorts were conducted. The first one was a retrospective evaluation of PTEN in two cohorts. (1) Archived formalin-fixed and paraffin-embedded brain metastasis specimens (n = 131) from patients with a history of breast cancer who presented with metastasis to the brain parenchyma and had surgery at the MDACC (Supplementary Information). Tissues were collected under a protocol (LAB 02-486) approved by the Institutional Review Board (IRB) at the MDACC. (2) Archived unpaired primary breast cancer formalin-fixed and paraffin-embedded specimens (n = 139) collected under an IRB protocol (LAB 02-312) at the MDACC (Supplementary information). Formal consent was obtained from all patients. The second study was a retrospective evaluation of PTEN, CCL2 and IBA1 in the matched primary breast tumours and brain metastatic samples from 35 patients, of which there are 12 HER2-positive, 14 triple-negative and nine oestrogen-receptor-positive tumours according to clinical diagnostic criteria (Supplementary Information). Formalin-fixed, paraffin-embedded primary breast and metastatic brain tumour samples were obtained from the Pathology Department, University of Queensland Centre for Clinical Research. Tissues were collected with approval by human research ethics committees at the Royal Brisbane and Women’s Hospital (2005/022) and the University of Queensland (2005000785). For tissue microarray construction, tumour-rich regions (guided by histological review) from each case were sampled using 1-mm cores. All the archival paraffin-embedded tumour samples were coded with no patient identifiers. Standard IHC staining was performed as described previously28. In brief, after de-paraffinization and rehydration, 4 μm sections were subjected to heat-induced epitope retrieval (0.01 M citrate for PTEN). Slides were then incubated with various primary antibodies at 4 °C overnight, after blocking with 1% goat serum. Slides underwent colour development with DAB and haematoxylin counterstaining. Ten visual fields from different areas of each tumour were evaluated by two pathologists independently (blinded to experiment groups). Positive IBA1 and Ki-67 staining in mouse tumours were calculated as the percentage of positive cells per field (%) and normalized by the total cancer cell number in each field. TUNEL staining was counted as the average number of positive cells per field (10 random fields). We excluded necrotic areas in the tumours from evaluation. Immunofluorescence was performed following the standard protocol recommended by Cell Signaling. In brief, after washing with PBS twice, cells were fixed with 4% formaldehyde. Samples were blocked with 5% normal goat serum in PBS for 1 h before incubation with a primary antibody cocktail overnight at 4 °C, washed, then incubated with secondary antibodies before examination using confocal microscope. Pathologists were blinded to the group allocation during the experiment and when assessing the outcome. Publicly available GEO data sets GSE14020, GSE19184, GSE2603, GSE2034 and GSE12276 were used for bioinformatics analysis. The top 2 × 104 verified probes were subjected to analysis. Differentially expressed genes between metastases from brain and other sites (primary or other metastatic organ sites) were analysed by SAM analysis in R statistical software. The 54 commonly downregulated genes in brain metastases from GSE14020 and GSE19184 were depicted as a heat-map by Java Treeview. For staining of patient samples, we calculated the correlation by Fisher’s exact test. For survival analysis of GSE2603, the patient samples were mathematically separated into PTEN-low and -normal groups based on K-means (K = 2). Kaplan–Meier survival curves were generated by survival package in R. Multiple group IHC scores were compared by Chi-square test and Mantelhaen test in R. All quantitative experiments have been repeated using at least three independent biological repeats and are presented as mean ± s.e.m. or mean ± s.d.. Quantitative data were analysed either by one-way analysis of variance (ANOVA) (multiple groups) or t-test (two groups). P < 0.05 (two-sided) was considered statistically significant.
News Article | October 27, 2016
Dublin, Oct. 27, 2016 (GLOBE NEWSWIRE) -- Research and Markets has announced the addition of the "Genome Editing Global Market-Forecast to 2022" report to their offering. The genome editing global market over the forecast period 2015 to 2022 and the market is expected to grow at a CAGR of 31.1%. The Genome editing global market is segmented based on technology, applications, products, end users and geography. Technologies segment consists of Zinc Finger Nucleases (ZFN), Transcriptor-activator-like effector nuclease (TALEN); Clustered regularly interspaced short palindromic repeats (CRISPR) and other gene modification techniques such as Recombinant adeno-associated virus (rAAV), piggyBac transposon, megatales etc. Applications identified includes, basic research, agriculture biotechnology, animal research, drug discovery and development. Products are classified into reagents, enzymes and consumables, instruments, cell lines & animal models and software. Depending on the end users, the genome editing global market is sectioned into academic & government institutions, pharmaceutical & biotechnology companies, plant biotechnology and others. Increased R&D expenditure and growth of biotechnology and pharmaceutical industries, increasing private and public sector funding, rapid advancements in sequencing and gene editing technologies, non-labeling of gene-edited products as Genetically Modified Organisms (GMOs), applications in various drug discovery processes are some of the factors driving the genome editing global market growth. Factors such as stringent regulatory framework, ethical issues concerning editing human embryo and adverse public perception, unavailability of gene-editing based therapeutics in the market, off-target effects of CRISPR and patent disputes associated with CRISPR technology are hampering the market growth. The genome editing global market is a consolidated market with key players such as Applied Stemcell (U.S.), Cellectis S.A. (France), Genscript (U.S.), Horizon Discovery Group (U.K.), Merck KGaA (Germany), Origene Technologies (U.S.), Sangamo Biosciences (U.S.), System Biosciences (U.S.), Thermo Fisher Scientific (U,S,), Transposagen (U.S.). Key Topics Covered: 1 Executive Summary 2 Introduction 2.1 Key Takeaways 2.2 Report Description 2.3 Markets Covered 2.4 Stakeholders 2.5 Research Methodology 3 Market Overview 3.1 Introduction 3.2 Market Segmentation 3.3 Factors Influencing Market 3.4 Market Dynamics 3.4.1 Drivers And Opportunities 188.8.131.52 Increased R&D Expenditure And GROWth Of Biotechnology And Pharmaceutical Industries. 184.108.40.206 Investments From Both Public And Private Sectors For Gene-Editing Technology 220.127.116.11 Technological Advancements In Sequencing And Gene Editing Technologies 18.104.22.168 Non-Transgenic Breeding Technologies And Gene-Edited Plants Not Labelled As Gmo In Many Countries 22.214.171.124 Improvement In Drug Discovery Process 126.96.36.199 Varied Applications In Drug Discovery And Development, Plant Engineering, Improvement In Animal Traits, Therapeutics 188.8.131.52 Precision Medicine And New Therapeutics For Genetic And Other Disorders 3.4.2 Restraints And Threats 184.108.40.206 Stringent Regulatory Frameworks 220.127.116.11 Ethical Issues Concerning Editing Human Embryo And Adverse Public Perception 18.104.22.168 Unavailability Of Gene-Editing Based Therapeutic Product In The Market And Less Geographic Penetration Due To Uncertain Regulations 22.214.171.124 Patent Dispute Associated With Crispr/Cas 126.96.36.199 Off-Target Effects Of Crispr/Cas9 Genome Editing Technology 3.5 Porter's Five Force Analysis 3.6 Regulatory Affairs 3.7 Patent Analysis 3.8 Funding Scenario 3.9 Collaboration, Joint Venture, Partnership, Agreement 3.1 Market Share Analysis 4 Genome Editing Global Market, By Technology 4.1 Introduction 4.2 Zinc Finger Nuclease (Zfn) 4.3 Transcription Activator-Like Effector Nucleases (Talen) 4.4 Clustered Regularly Interspaced Short Palindromic Repeats (Crispr/Cas9) 4.4.1 Wt Crispr/Cas9 4.4.2 Crispr/Cas9 Nickase 4.4.3 Crispr Dcas9 4.5 Others 4.5.1 Recombinant Adeno-Associated Virus (R Aav) 4.5.2 Piggybac Transposase And Sleeping Beauty Transposon 4.5.3 Arcus (Homing Endonuclease) And Megatal 4.5.4 Targatt And Rapid Trait Development System (Rtds) 5 Genome Editing Global Market, By Application 5.1 Introduction 5.2 Basic Research 5.2.1 Transcription Activation/Repression 5.2.2 Genomic Screening 5.2.3 Genomic Visualization 5.3 Plant Biotechnology/Agriculture 5.4 Animal Biotechnology 5.4.1 Animal Health 5.4.2 Livestock 5.4.3 Other 5.5 Drug Discovery And Development 5.5.1 Pre-Clinical 5.5.2 Clinical 6 Genome Editing Global Market, By Products And Services 6.1 Introduction 6.2 Reagents, Enzymes And Consumables 6.2.1 Genome Editing Tools And Kits 6.2.2 Delivery Tools 6.2.3 Other Reagents, Enzymes And Consumables 6.3 Cell Lines And Animal Models 6.4 Genome Editing Services 6.5 Instruments 6.6 Software 7 Genome Editing Global Market, By End Users 7.1 Introduction 7.2 Academic And Government Research Institutes 7.3 Plant Biotechnology Companies 7.4 Pharmaceutical And Biotechnology Companies 7.5 Others 7.5.1 Animal Biotechnology 7.5.2 Contract Research Organizations (Cro) 8 Genome Editing Global Market, By Region 9 Competitive Landscape 9.1 Introduction 9.1.1 Licensing Agreement And Others As A Major GROWth Strategy Of Market Players 9.2 Licensing Agreement 9.3 Other Developments 9.4 Agreement/Collaboration/Partnership/Joint Venture 9.5 Approval 9.6 New Product Launch 9.7 Acquisitions 10 Major Companies - Abcam - Addgene - Agilent Technologies - Applied Biological Materials (Abm) - Axol Bioscience Ltd - Bio-Rad Laboratories - Caribou Biosciences - Charles River Laboratories - Cibus - Crispr Therapeutics - Desktop Genetics - Discovery Genomics - Editas Medicine - Eisai - Ers Genomics - GE Healthcare - Genecopoeia - Genus Inc - Homology Medicines - Illumina - Integrated Dna Technologies (Idt) - Intellia Therapeutics - Lonza - New England Biolabs (Neb) - Pacific Bioscience - Precision Biosciences - Proteonic B.V. - Recombinetics - Shire Plc - Stemgent, Inc - Syngenta - Takara Bio - Targetgene Biotechnologies - Tide - Toolgen - Transgenic - Viravecs Labs For more information about this report visit http://www.researchandmarkets.com/research/c2g57b/genome_editing
News Article | February 22, 2017
No statistical methods were used to predetermine sample size. The experiments were not randomized and the investigators were not blinded to allocation during experiments and outcome assessment. Animal experiments were neither randomized nor blinded. All animals used in this study (ADicerKO, Dicerlox/lox, or C57BL/6) were males and 6 months of age unless specifically indicated otherwise. All animal experiments were conducted in accordance with IRB protocols with respect to live vertebrate experimentation. Human serum was obtained from human subjects after obtaining IRB approval and patients were given informed consent. Mouse and human sera were centrifuged at 1,000g for 5 min and then at 10,000g for 10 min to remove whole cells, cell debris and aggregates. The serum was subjected to 0.1-μm filtration and ultracentrifuged at 100,000g for 1 h. Pelleted vesicles were suspended in 1× PBS, ultracentrifuged again at 100,000g for washing, resuspended in 1× PBS and prepared for electron microscopy and immune-electron microscopy or miRNA extraction. All in vitro experiments were carried out using exosome-free FBS. AML-12 cells were acquired from ATCC (cat no. CRL-2254) and were tested for mycoplasma contamination. Dicerfl/fl brown preadipocytes were generated as previously described16. For exosome loading, exosome preparations were isolated and diluted with PBS to final volume of 100 μl. Exosome electroporation was carried out by using a variation of a previously described technique45. Exosome preparations were mixed with 200 μl phosphate-buffered sucrose (272 mM sucrose 7 mM, K HPO ) with 10 nΜ of a miRNA mimic, and the mixture was pulsed at 500 mV with 250 μF capacitance using a Bio-Rad Gene Pulser (Bio-Rad). Electroporated exosomes were resuspended in a total volume of 500 μl PBS and added to the target cells. Isolated exosomes were subjected to immune-electron microscopy using standard techniques7. In brief, exosome suspensions were fixed with 2% glutaraldehyde supplemented with 0.15 M sodium cacodylate and post-fixed with 1% OsO . They were then dehydrated with ethanol and embedded in Epon 812. Samples were sectioned, post-stained with uranyl acetate and lead citrate, and examined with an electron microscope. For immune-electron microscopy, cells were fixed with a solution of 4% paraformaldehyde, 2% glutaraldehyde and 0.15 M sodium cacodylate and processed as above. Sections were probed with anti-CD63 (Santa Cruz Biotechnology, sc15363) or anti-CD9 (Abcam, ab92726) antibodies (or rabbit IgG as a control) and visualized with immunogold-labelled secondary antibodies. Immuno-electron microscopy analysis revealed that the isolated exosomes were 50–200 nM in diameter and stained positively for the tetraspanin exosome markers CD63 and CD9 (ref. 46). Exosomal concentration was assessed using the EXOCET ELISA assay (System Biosciences), which measured the esterase activity of cholesteryl ester transfer protein (CETP) activity. CETP is known to be enriched in exosomal membranes. The assay was calibrated using a known isolated exosome preparation (System Biosciences). Additionally, exosome preparations were subjected to the qNano system employing tunable resistive pulse sensing technology (IZON technologies) to measure the number and size distribution of exosomes. For total serum miRNA isolation, 100 μl of serum was obtained from ADicerKO mice or Lox littermates and miRNAs were isolated using an Exiqon miRCURY Biofluid RNA isolation kit following the manufacturer’s protocol. RNA was isolated from exosomal preparations using TRIzol, following the manufacturer’s protocol (Life Sciences). Subsequently, 50 ng of exosomal RNA was subjected to reverse transcription into cDNA by using a mouse miRNome profiler kit (System Biosciences). qPCR was then performed in 6-μl reaction volumes containing cDNA along with universal primers for each miRNA and SYBR Green PCR master mix (Bio-Rad). In line with previous research, for all serum and exosomal miRNA quantitative PCR reactions the C values were normalized using U6 snRNA as an internal control. To estimate miRNA abundance in fat tissue, data were normalized using the global average of expressed C values per sample47, as the snRNA U6 was differentially expressed between depots. For all quantitative PCR reactions involving gene expression calculations for FGF21, normalization was carried out by using the TATA-binding protein as an internal control. Differential expression analysis of the high-throughput −ΔC values was done using the Bioconductor limma package48 in R (www.r-project.org). Fold differences in comparisons were expressed as 2−ΔΔC . Ct Principal component analysis plots were created using R with the ggplot2 package. A detection threshold was set to C = 34 for all mouse miRNA PCR reactions; no threshold was used for human miRNA PCR, according to the manufacturer’s recommendation (System Biosciences). An miRNA was plotted only if its raw C value was ≤34 in at least three samples, except for the brown pre-adipocyte and 4-week-old mouse experiments, in which the raw C only had to be ≤34 twice. miRNA −ΔC values were Z scored and heatmaps were created by Cluster 3.0 and TreeView programs as previously described49. Fat tissue transplantation was carried out as previously described50. In brief, 10-week-old male Lox donor mice (C57BL/6 males) were killed, and their inguinal, epididymal, and BAT fat depots were isolated, cut into several 20-mg pieces and transplanted into 10-week-old male ADicerKO mice (n = 5 male mice per group). Each recipient ADicerKO mice received the equivalent transplanted fat mass of two donor Lox control mice. Transplanted mice received post-surgical analgesic intraperitoneal injections (buprenorphine, 50 mg/kg) for 7 days. At day 12, a glucose tolerance test was performed after a 16-h fast by intraperitoneal injection of 2 g/kg glucose. All mice were killed after 14 days. All procedures were conducted in accordance with Institutional Animal Care and Use Committee regulations. An adenoviral Fgf21 3′ UTR reporter was created by cloning the 3′ UTR of Fgf21 into the pMir-Report vector. Subsequently, the luciferase-tagged 3′ UTR fragment was cloned into the adenoviral vector pacAd5-CMV-IRES-GFP, creating an adenovirus bearing the Fgf21 3′UTR reporter. Hsa-miR-302f-3′-UTR was created by cloning the synthesized Luc-miR-302f-3′-UTR fragment (Genescript) into the Viral Power Adenoviral Expression System (Invitrogen). In vitro bioluminescence was measured using a dual luciferase kit (Promega). Eight-week-old male ADicerKO or wild-type mice were injected intravenously with adenovirus bearing the 3′-UTR of Fgf21 fused to the luciferase gene to create two groups of liver-reporter mice—one with a ADicerKO background and one with a wild-type background. One day later, a third group of ADicerKO mice, which had also been injected intravenously with an adenovirus bearing the 3′-UTR of Fgf21 fused to the luciferase gene, were injected intravenously with exosomes isolated from the serum of wild-type mice. After 24 h, in vivo luminescence of the Fgf21 3′ UTR was measured using the IVIS imaging system (Perkin Elmer) by administering d-luciferin (20 mg/kg) according to the manufacturer’s protocol (Perkin Elmer). For the second group, 8-week-old male ADicerKO or wild-type mice were also transfected with adenovirus bearing the 3′ UTR of Fgf21 fused to the luciferase gene by intravenous injection. After 1 day, mice received an intravenous injection of exosomes isolated from the serum of either ADicerKO mice or ADicerKO mice reconstituted in vitro with 10 nM miR-99b by electroporation. Twenty-four hours later, in vivo luminescence was measured using the IVIS imaging system by administering d-luciferin (20 mg/kg) according to the manufacturer’s protocol (Perkin Elmer). Protocol 1. On day 0, adenovirus bearing either pre-hsa_miR-302f or lacZ mRNA (as a control) was injected directly into the BAT of 8-week-old male C57BL/6 mice. Hsa_miR-302f is human-specific and does not have a mouse homologue. This procedure was conducted under ketamine-induced anaesthesia. Four days later, the same mice were injected intravenously with an adenovirus bearing the 3′ UTR of miR-302f in-frame with the luciferase gene, thereby transducing the liver tissue of the mouse with this human 3′ UTR miRNA reporter. Suppression of the 3′ UTR miR-302f reporter would occur only if there was communication between the BAT-produced miRNA and the liver. In vivo luminescence was measured on day 6 using the IVIS imaging system as described above. Protocol 2. To assess specifically the role of exosomal miR-302f in the regulation of its target reporter in the liver, two separate cohorts of 8-week-old male C57BL/6 mice were generated. One cohort was injected with adenovirus bearing pre-miR-302f or lacZ directly into the BAT (the donor cohort); the second cohort was transfected with an adenovirus bearing the 3′ UTR reporter of this miR-302f in the liver, as described for Protocol 1 (the acceptor cohort). Serum was obtained on days 3 and 6 from the donor cohorts; the exosomes were isolated and then injected intravenously into the acceptor mice the next day (days 4 and 7). On day 8, in vivo luminescence was measured in the acceptor mice using the IVIS imaging system as described above. To test for the presence of adenovirus in the liver and BAT of C57BL/6 mice, 100 mg tissue was homogenized in 1 ml sterile 1× PBS. The homogenate was spun down and 150 μl cleared supernatant was used to isolate adenoviral DNA using the Nucleospin RNA and DNA Virus kit, following the manufacturer’s protocol (Takara). PCR was performed on 2 μl isolated adenoviral DNA using SYBR green to detect lacZ or miR-302f amplicons. For all PCR data obtained in the fat-tissue-transplantation experiment, an miRNA was considered to be present only if its mean C in the wild-type group was <34. We then identified those miRNAs that were significantly decreased in ADicerKO serum. For an miRNA to be considered restored after transplantation by a particular depot it had to be significantly increased from ADicerKO serum with a mean C < 34 and its C had to be more than 50% of the way from ADicerKO to the wild type on the C scale. ANOVA tests were followed by two-tailed Dunn’s post-hoc analysis or Tukey’s multiple comparisons test to identify statistically significant comparisons. All t-tests and Mann–Whitney U-tests were two-tailed. P values less than 0.05 were considered significant. All ANOVA, t-tests, and area-under-the-curve calculations were carried out in GraphPad Prism 5.0. For miRDB analysis (http://www.mirdb.org), a search by target gene was performed against the mouse database. A target score of 85 was set to exclude potential false-positive interacting miRNAs. All high-throughput qRT–PCR data (raw C values), the code used to analyse them (in the free statistical software R), and its output (including supplementary tables, tables used to generate heatmaps, and statements in the text) can be freely downloaded and reproduced from https://github.com/jdreyf/fat-exosome-microrna. All other data are available from the corresponding author upon reasonable request.
Razvi E.S.,System Biosciences
Drugs of the Future | Year: 2013
Exosomes/microvesicles (EMVs) offer significant promise for the development of liquid biopsies for a number of disease classes, most notably cancer. The current challenges in the isolation and purification of EMVs are slowly being addressed by new technologies aimed at the research marketplace, as well as the downstream diagnostics market. Once isolated and purified, the challenge for the study of these vesicles moves towards interrogation of the cargo (e.g., RNA) and proteins contained therein. There is the need to "associate" specific molecular signatures with specific clinical sequelae. This is perhaps the most intractable problem and will require significant investment of resources and time to establish these relationships. Additionally, circulating tumor cells represent yet another class of circulating biomarkers, and it is believed that different types of circulating biomarkers will be deployed in liquid biopsies for different disease classes. Over the coming decade the marketplace will be segmented by disease classes first and technologies/ biomarker classes second. However, the inescapable conclusion is that the era of minimally invasive biopsies is coming closer, and will provide less painful procedures, better manage ment and cost-effective diagnostics. © 2013 Prous Science, S.A.U. or its licensors.
Razvi E.S.,System Biosciences
Regenerative Medicine | Year: 2011
The Cambridge Healthtech Institutes (CHI) 18th International Molecular Medicine Tri-Conference 2011 brought together all the key stakeholders in the stem cells marketplace including academic researchers, researchers from biotechnology companies, as well as business leaders involved in various aspects of the regenerative medicine space. The focus of the stem cells track, of this 12-track conference, was on the translation of basic stem cell discoveries into preclinical and clinical milestones. Many presentations focused on the production of induced pluripotent stem cells from various patient populations and the potential utilization of induced pluripotent stem cells in the future for cellular therapy. In this article, I provide a snapshot of this conference and highlight selected presentations, giving the overall direction that the stem cell field is taking. © 2011 Future Medicine Ltd.
Ho T.-T.,University of Mississippi Medical Center |
Zhou N.,University of Mississippi Medical Center |
Huang J.,University of Mississippi Medical Center |
Koirala P.,University of Mississippi Medical Center |
And 4 more authors.
Nucleic Acids Research | Year: 2015
The CRISPR/Cas has been recently shown to be a powerful genome-editing tool in a variety of organisms. However, these studies are mainly focused on protein-coding genes. The present study aims to determine whether this technology can be applied to non-coding genes. One of the challenges for knockout of non-coding genes is that a small deletion or insertion generated by the standard CRISPR/Cas system may not necessarily lead to functional loss of a given non-coding gene because of lacking an open reading frame, especially in polyploidy human cell lines. To overcome this challenge, we adopt a selection system that allows for marker genes to integrate into the genome through homologous recombination (HR). Moreover, we construct a dual guide RNA vector that can make two cuts simultaneously at designated sites such that a large fragment can be deleted. With these approaches, we are able to successfully generate knockouts for miR-21, miR-29a, lncRNA-21A, UCA1 and AK023948 in various human cell lines. Finally, we show that the HR-mediated targeting efficiency can be further improved by suppression of the non-homologous end joining pathway. Together, these results demonstrate the feasibility of knockout for non-coding genes by the CRISPR/Cas system in human cell lines. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.
Liu Q.,University of Illinois at Springfield |
Liu Q.,Chongqing Medical University |
Huang J.,University of Illinois at Springfield |
Huang J.,University of Mississippi Medical Center |
And 8 more authors.
Nucleic Acids Research | Year: 2013
Protein-coding genes account for only a small part of the human genome, whereas the vast majority of transcripts make up the non-coding RNAs including long non-coding RNAs (lncRNAs). Accumulating evidence indicates that lncRNAs could play a critical role in regulation of cellular processes such as cell growth and apoptosis as well as cancer progression and metastasis. LncRNA loc285194 was previously shown to be within a tumor suppressor unit in osteosarcoma and to suppress tumor cell growth. However, it is unknown regarding the regulation of loc285194. Moreover, the underlying mechanism by which loc285194 functions as a potential tumor suppressor is elusive. In this study, we show that loc285194 is a p53 transcription target; ectopic expression of loc285194 inhibits tumor cell growth both in vitro and in vivo. Through deletion analysis, we identify an active region responsible for tumor cell growth inhibition within exon 4, which harbors two miR-211 binding sites. Importantly, this loc285194-mediated growth inhibition is in part due to specific suppression of miR-211. We further demonstrate a reciprocal repression between loc285194 and miR-211; in contrast to loc285194, miR-211 promotes cell growth. Finally, we detect downregulation of loc285194 in colon cancer specimens by quantitative PCR arrays and in situ hybridization of tissue microarrays. Together, these results suggest that loc285194 is a p53-regulated tumor suppressor, which acts in part through repression of miR-211. © 2013 The Author(s).