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Gene Bridges GmbH | Date: 2017-03-08

A method for performing homologous recombination between at least a first nucleic acid molecule and a second nucleic acid molecule which share at least one region of sequence homology. A method for improving the efficiency of homologous recombination.

News Article | November 16, 2016
Site: www.nature.com

Male C57BL/6J mice (CLEA Japan) were treated with ethylnitrosourea (85 mg kg−1, Sigma-Aldrich) by intraperitoneal injection twice at weekly intervals at the age of 8 weeks. At the age of 25–30 weeks, the sperm of the mice was used for in vitro fertilization with eggs of C57BL/6N mice to obtain F offspring. Mice were provided food and water ad libitum, and were maintained on a 12-h light:12-h dark cycle and housed under controlled temperature and humidity conditions. All procedures were approved by the Institutional Animal Care and Use Committee of the University of Tsukuba and the RIKEN BioResource Center, University of Texas Southwestern Medical Center at Dallas. EEG/EMG electrode implantation was performed as described previously35, with isoflurane (3% for induction, 1% for maintenance) used for anaesthesia. Seven days after surgery, the mice were tethered to a counterbalanced arm (Instech Laboratories) that allowed free movement and exerted minimal weight. At the age of 12 weeks, male mice were implanted with EEG/EMG electrodes and then screened for sleep/wakefulness behaviour. Examined parameters were total time spent in wake, NREMS and REMS states, episode duration of wake, NREMS and REMS states, appearance of muscle atonia during REMS, and rebound sleep after 4-h sleep deprivation by shaking the cages. For quantitative parameters, we selected mice whose phenotypes deviated from the average by at least 3 standard deviations. After confirming the reproducibility of the sleep phenotype, the mice were selected for offspring production by natural mating or IVF with wild-type females to examine the heritability of the sleep phenotypes. If at least 30% of the male littermates showed sleep phenotypes similar to their father, we considered the sleep abnormalities to be heritable. Single nucleotide polymorphisms of N mice were determined using a custom TaqMan Genotyping assay (Thermo Fisher). The custom probes were designed based on the polymorphism data between C57BL/6J and C57BL/6N (ref. 20). QTL analysis was performed using J/qtl software (Jackson Laboratory). Whole exomes were captured with SureSelectXT2 Mouse All Exon (Agilent) and processed to a paired end 2 × 100-bp run on the Illumina HiSeq2000 platform at the UTSW McDermott Center Next Generation Sequencing Core. Reads were mapped to the University of California Santa Cruz mm9 genome reference sequence for C57BL/6J using Burrows–Wheeler aligner and quality filtered using SAMtools. Cleaned BAM files were used to realign data and call variants using the Genome Analysis ToolKit to detect heterozygous mutations. The recording room was kept under 12-h light:12-h dark cycles and a constant temperature (24–25 °C). To examine sleep–wake behaviour under baseline conditions, EEG/EMG signals were recorded for two consecutive days from the onset of the light phase. EEG/EMG data were visualized and analysed using a MatLab (MathWorks)-based, custom semi-automated staging program followed by visual inspection. EEG signals were subjected to fast Fourier transform analysis from 1 to 30 Hz with 1-Hz bin using MatLab-based custom software. Epochs containing movement artefacts were included in the state totals but excluded from subsequent spectral analysis. Sleep/wakefulness was staged into wakefulness, NREMS and REMS. Wakefulness was scored based on the presence of low amplitude, fast EEG, and high amplitude, variable EMG. NREMS was characterized by high amplitude, delta (1–4 Hz) frequency EEG and low EMG tonus, whereas REMS was staged based on theta (6–9 Hz)-dominant EEG and EMG atonia. Hourly delta density during NREMS indicates hourly averages of delta density which is the ratio of delta power to total EEG power at each 20-s epoch. For the power spectrum of sleep/wakefulness, the EEG power of each frequency bins was expressed as a percentage of the total EEG power over all frequency bins (1–30 Hz) and sleep/wakefulness states35, 36. For sleep deprivation, mice were sleep deprived for 2, 4 and 6 h from the onset of the light phase by gently touching the cages when they started to recline and lower their heads. Food and water were available. To evaluate the effect of sleep deprivation, the NREMS delta power during the first hour after sleep deprivation was expressed relative to the same zeitgeber time (ZT) of the basal recording or relative to the mean of the basal recording. For caffeine and modafinil injection experiments, mice were fully acclimatized for intraperitoneal injection before sleep recording. After 24-h baseline recording, mice received caffeine (Sigma), modafinil (Sigma) or vehicle (0.5% methyl cellulose (Wako)) intraperitoneally at ZT0, followed by 12-h recording. Injections were delivered once per week, with each injection followed by a 6–8-day washout period, during which mice remained in the recording chamber. To examine the sleep/wakefulness behaviour under constant darkness, after 48-h recording under a 12-h light:12-h dark cycle, EEG/EMG recording continued in constant darkness for 3 days. Mice were housed individually in a cage (width 23 cm, length 33 cm, height 14 cm) containing a wireless running wheel (Med Associate ENV-044). Cages were placed in a light-tight chamber equipped with green LED light (100 lx at the bottom of the cage). The rotation numbers of wheels were obtained with 1-min bin using Wheel manager software (Med Associate). Mice were entrained to 12-h light:12-h dark cycle for 7 days, and then released into constant darkness for 3 weeks. The free running period was calculated with linear regression analysis of activity onset using MatLab-based custom software. Circadian activity amplitude was calculated by fast Fourier transform of activity data, which were processed with Bartlett window using MatLab-based custom software. Relative amplitude was normalized to the mean amplitude of the wild- type group. A rabbit polyclonal antibody against the C-terminal 171 amino acids of mouse SIK3 was generated using custom antibody production service (Pacific Immunology). Tissues were homogenized using a rotor-stator homogenizer (Polytron) in ice-cold lysis buffer (20 mM HEPES, pH 7.5, 100 mM NaCl, 10 mM Na P O , 1.5% Triton X-100,15 mM NaF, 1× PhosSTOP (Roche), 5 mM EDTA, 1× protease inhibitor (Roche)), and then centrifuged at 13,000g at 4 °C. The supernatants were separated by SDS–PAGE and transferred on PVDF membrane. Western blotting was performed according to standard protocols. In situ hybridization was performed as described previously37. In brief, a 0.7–0.8-kb fragment of Nalcn cDNA was inserted into pGEM-T easy (Promega) and used for DIG-labelled probe synthesis. Mice were deeply anaesthetized with sodium pentobarbital and perfused transcardially with PBS followed by 4% paraformaldehyde (PFA). Forty-micrometre-thick brain sections were treated with 0.3% Triton X-100, digested with 1 μg ml−1 proteinase K, treated with 0.75% glycine, and then treated with 0.25% acetic anhydride in 0.1 M triethanolamine. After overnight incubation with a digoxigenin (DIG)-labelled probe at 60 °C, the sections were washed and then incubated with alkaline phosphatase-conjugated anti-DIG Fab fragments (Roche, 11175041910). The reactions were visualized with a 5-bromo-4-chloro-3-indolyl-phosphate/4-nitroblue tetrazolium (BCIP/NBT) substrate solution (Roche). HEK293 cells (RCB1637) and HEK293T cells (RCB2202) were obtained from the RIKEN BRC Cell Bank. Cells were cultured in DMEM (Wako) supplemented with 10% FBS, 1% GlutaMAX (Thermo Fisher Scientific), and penicillin/streptomycin at 37 °C in a humidified atmosphere of 5% CO . Cell lines were regularly tested for mycoplasma contamination using MycoAlert (Lonza). Cell lines were regularly renewed by obtaining cell stocks from the Cell Bank for authentication. We used HEK293 and HEK293T cells because of their reliable growth, high efficiency in transfection and morphology suitable for electrophysiological experiments. For generating Sik3Slp knock-in mice, a genomic fragment containing exon 13 of the Sik3 gene was isolated from C57BL/6 mouse genomic BAC clone from a RP23 mouse genomic BAC library (Advanced GenoTEchs Co). A 1.7-kb fragment of FRT-PGK-gb2-neo-FRT-loxP cassette (Gene Bridges) flanked by two flippase recognition target (FRT) sites was inserted before exon 12. The targeting vector also contains a G-to-A substitution at the fifth nucleotide from the beginning of intron 13. The targeting vector was linearized and electroporated into the C57BL/6N ES cell line RENKA. Correctly targeted clones were injected into eight-cell stage ICR mouse embryos, which were cultured to produce blastocysts and then transferred to pseudopregnant ICR females. Resulting male chimaeric mice were crossed with female C57BL/6N mice to establish the Sik3Slp-neo/+ line. To remove the neomycin resistance gene with the FLP-FRT system, Sik3Slp-neo/+ mice were crossed with Actb-FLP knock-in mice. The custom-designed ZFN mRNAs targeting the exon 13–intron 13 boundary region of the Sik3 gene were obtained from Sigma-Aldrich’s Composers Custom ZFN service. Before the final assembly of the ZFN products, Sigma-Aldrich validated the designed ZFN binding sequences in silico using their bioinformatics tools and in vitro using Nero2A cell lines, ensuring high cutting efficiency and specificity using mismatch-specific endonuclease CelI according to the manufacturer’s instructions. The ZFN mRNAs were injected into single-cell stage C57BL/6J mouse zygotes at the University of Texas Southwestern Transgenic Core facility. The injected eggs were then transferred to pseudopregnant females to generate F founders. In total, 45 out of 96 F mice were found to be modified at the exon 13–intron 13 boundary region of the Sik3 gene. We crossed one F male mouse that had a 2-bp deletion from the last nucleotide of exon 13 with female C57BL/6N mice to obtain F mice of Sik3Slp/+ ZFN. The F mice were used to confirm the skipping of exon13 in Sik3 mRNA, which was purified from the brains and livers. The F male mice were used for sleep/wakefulness behaviour analysis. To produce a Cas9/single-guide RNA (sgRNA) expression vector, oligonucleotide DNAs (5′-CACCGCGAGCGGCCATCGACCCGC-3′ and 5′-AAACGCGGGTCGATGGCCGCTCGC-3′) were annealed and then inserted into pX330 vector (Addgene). The cleavage activity of the pX330-Sik3Ex1 vector was evaluated by the EGxxFP system38. Genomic DNA containing exon 1 of the Sik3 gene was amplified and inserted into pCAG-EGxxFP to produce pCAG-EGxxFP-Sik3Ex1. The pX330-Sik3Ex1 and pCAG-EGxxFP-Sik3Ex1 were transfected into HEK293 cells. As a donor oligonucleotide, a single-stranded 200-nucleotide DNA was synthesized (Integrated DNA Technologies), which contained a Flag-haemagglutinin-coding sequence in the centre and 70-nucleotide arms at the 5′ and 3′ ends. Female C57BL/6J mice or Sik3Slp knock-in mice were injected with pregnant mare serum gonadotropin and human chorionic gonadotropin at a 48-h interval, and mated with male C57BL/6J mice. The fertilized one-cell embryos were collected from the oviducts. Then, 5 ng μl−1 of pX330-Sik3Ex1 vector and 10 ng μl−1 of the donor oligonucleotide were injected into the pronuclei of these one-cell-stage embryos. The injected one-cell embryos were then transferred into pseudopregnant ICR mice. F mice were genotyped for the presence of Flag-coding sequence in exon1 of the Sik3 gene and for the presence of the Sik3Slp mutation. F mice containing Flag–SIK3 were further examined for the presence of the Cas9 transgene and off-target effects. Candidate off-target sites were identified based on a complete match of 16 bp at the 3′ end, including the PAM sequence. F mice were mated with C57BL/6N mice to obtain F offspring. NalcnDrl mice were produced as described above. To produce the sgRNA expression vector, pX330-NalcnEx9, oligonucleotide DNAs (5′-CACCAGCAATAAACACATTCTGAA-3′ and 5′-AAACTTCAGAATGTGTTTATTGCT-3′) were used. Genomic DNA containing exon 9 of the Nalcn gene was amplified and inserted into pCAG-EGxxFP to produce pCAG-EGxxFP-NalcnEx9. As a donor oligonucleotide, a single-stranded 199-nucleotide DNA containing a T-to-A substitution at the centre was synthesized (Integrated DNA Technologies). Nalcn mutant mice of N –N generation were used for sleep/wakefulness analysis. To evaluate Flag-tagged SIK3 protein in brains, we performed peptide mapping of the purified Flag–SIK3 protein. The brains of Flag-Sik3 knock-in mice and Flag-Sik3Slp knock-in mice were quickly dissected after cervical dislocation. Brains were homogenized in detergent-free buffer and then centrifuged (100,000g, 30 min, 4 °C). The supernatant was immunoprecipitated with anti-DDDDK antibody beads (MBL 3325). The eluate was run on a polyacrylamide gel and stained with SilverQuest Silver staining kit (Life technologies). Flag–SIK3 band (150 kDa) was dissected with a fresh blade. The proteins in the bands were reduced with 10 mM dithiothreitol and alkylated with 40 mM iodoacetamide. Each sample was digested with trypsin (4 μg ml−1; Trypsin Gold, Promega) at 37 °C overnight. The extracted peptides were then separated via nano flow LC (Advance LC, Michrom Bioresources) using a C18 column. The LC eluate was coupled to a nano-ionspray source attached to a Orbitrap Velos Pro mass spectrometer (Thermo Fisher Scientific). All MS/MS spectra were searched using Proteome Discoverer 1.3 software (Thermo Fisher Scientific). Peptides were mapped through mouse SIK3 (NP_081774) with 56% coverage. To examine the effect of sleep deprivation on the phosphorylation status of SIK3 protein, five Flag-Sik3 knock-in mice or five Flag-Sik3Slp knock-in mice were ad libitum slept (S) or sleep-deprived (SD) for 4 h by gentle handling immediately after light onset (ZT0–ZT4). Five wild-type mice were used as a negative control. At ZT4, mouse brains were quickly dissected after cervical dislocation, rinsed with cold PBS, and snap frozen in liquid nitrogen. Each half of the brains was lysed in 2 ml of ice-cold lysis buffer (20 mM HEPES, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 2 mM MgCl , 15 mM NaF, 10 mM Na P O ) freshly supplemented with protease/phosphatase inhibitor cocktail tablets (Roche), and homogenized in a glass tissue homogenizer. After brain homogenate was incubated for 30 min and centrifuged at 13,000g for 20 min at 4 °C, the supernatant was pre-cleared by IgG and Protein G beads for 30 min before immunoprecipitation. Each pre-cleared lysate was added to 50 μl of anti-Flag antibody-conjugated Sepharose beads (Sigma, A2220) and rotated overnight at 4 °C. After washing the beads five times with cold wash buffer (20 mM HEPES, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 2 mM MgCl , 15 mM NaF, 10 mM Na P O ), 50 μl of elution buffer (2% SDS, 60 mM Tris-HCl, pH 6.8, 50 mM DTT, 10% glycerol) was added and rotated for 10 min at 4 °C. Elution was repeated twice and combined into one eluate and analysed by western blotting. For each group of Flag knock-in mice, the five eluates of were mixed and equally split into two or three samples for mass spectrometric analysis. Thus, a total of six (Flag-Sik3) or nine (Flag-Sik3 and Flag-Sik3Slp) samples were reduced, alkylated, and trypsin digested overnight. After desalting, each sample was labelled with a different Tandem Mass Tag (TMT) reagent (Thermo Fisher Scientific), then all samples were combined into one mixture for HPLC fractionation using a C18 column. A total of 12 fractions were collected, and analysed separately on the Orbitrap-Fusion mass spectrometry platform (Thermo Fisher Scientific) using a reverse-phase liquid chromatography tandem mass spectrometry (LC–MS/MS) method. We performed data analysis to identify peptides and quantified reporter ion relative abundance using Proteome Discoverer 2.1 (Thermo Fisher Scientific). The relative abundance of quantified SIK3 phosphorylation sites was normalized with wild-type negative control and total SIK3 protein abundance. To express wild-type NALCN, we used pTracer-CMV2-ratNALCN-EF1α-EGFP (a gift from D. Ren)39. A single nucleotide substitution was induced to make pTracer-CMV2-ratNALCN(DRL)-EF1α-EGFP using a KOD plus Mutagenesis kit (Toyobo). HEK293T cells were grown to ~50% confluency in 12-well plates. Using Lipofectamine LTX (2 μl) and PLUS (1 μl) reagents (Thermo Fisher Scientific), the cells were cotransfected with 0.3 μg of each plasmid DNA encoding rat NALCN-EGFP (wild type or DRL), mouse UNC-80, and mouse SRC (Y529F) (constitutively active Src) in 12-well plates. UNC-80 and SRC kinase activate NALCN27, 28. In some experiments, the cells were incubated with 10 μM Gd3+ to inhibit NALCN. The cells were dissociated and plated on 18-mm coverslips coated with poly-l-lysine in fresh culture medium before patch-clamp recordings. All patch-clamp recordings from HEK293T cells were performed >72 h after transfection. Recording patch pipettes were pulled from glass capillaries (1B150F-4, World Precision Instruments) using a micropipette puller (P-97, Sutter Instrument) to give a resistance of ~9 MΩ. The series resistance of whole-cell recordings was ~40 MΩ, which was not compensated. Patch pipettes were filled with solution containing 150 mM CsOH, 120 mM methanesulfonic acid, 10 mM NaCl, 10 mM EGTA, 2 mM Mg ATP and 10 mM HEPES (pH 7.4 adjusted with methanesulfonic acid; osmolarity, 290−299 mOsm l−1 adjusted with CsCl). The cells on coverslips were transferred to a recording chamber under a fluorescence upright microscope (Axio Examiner D1, Zeiss) and continuously perfused with the bath solutions containing 150 mM NaCl, 3.5 mM KCl, 10 mM HEPES, 20 mM glucose, 5 mM NaOH, 2 mM MgCl and 1.2 mM CaCl (pH 7.4 adjusted; osmolarity, 300−310 mOsm l−1). The transfected cells were identified by enhance green fluorescent protein (eGFP) fluorescence. Patch-clamp recordings were performed at room temperature (24 °C) using a computer-controlled amplifier (MultiClamp 700B, Molecular Devices). The signals were digitized with A/D converter (Digidata 1440A, Molecular Devices), and acquired with Clampex (Molecular Devices) at a sampling rate of 50 kHz, and low-pass filtered at 5 kHz. At the end of recording, Gd3+ (10 μM) was used to confirm that the whole-cell currents were mediated through NALCN39. Data were analysed using Clampfit (Molecular Devices). The equilibrium potentials were calculated from I–V curves. Mean membrane conductance was estimated from the regression lines fitted to I–V curves from individual cells. Current, membrane conductance and charge transfer were normalized to membrane capacitance. Patch pipettes and recording system were the same as those used in recordings from HEK293 cells. Acute brain slices containing the DpMe were prepared from post-natal day 12–23 Nalcn+/+ or NalcnDrl/+ mice. After the induction of deep anaesthesia with isoflurane, mice were decapitated and the brains were rapidly removed into an ice-cold cutting solution containing 2.5 mM KCl, 1.25 mM NaH PO , 26 mM NaHCO , 25 mM glucose, 185 mM sucrose, 0.5 mM CaCl and 10 mM MgCl (pH 7.4, when bubbled with 95% O and 5% CO ). The brains were cut coronally into 200–250 μm-thick slices with a vibratome (VT-1200S, Leica). The slices were incubated at 37 °C for 1 h in artificial cerebrospinal fluid (aCSF) containing 125 mM NaCl, 2.5 mM KCl, 1.25 mM NaH PO , 26 mM NaHCO , 10 mM glucose, 2 mM CaCl and 1 mM MgCl (pH 7.4, when bubbled with 95% O and 5% CO ) before recordings. Slices were transferred to a recording chamber perfused with aCSF under an upright microscope (Axio Examiner D1, Zeiss). Patch pipettes were filled with solution containing 125 mM K-gluconate, 10 mM KCl, 10 mM HEPES, 0.5 mM EGTA, 8 mM phosphocreatine-Na , 4 mM ATP-Mg and 0.3 mM GTP-Na (pH 7.3 adjusted with KOH; osmolality, 290 mOsm l−1). The DpMe was identified with axon bundles. Recordings were made from cells located in the medial part of the DpMe. Cells showing no action potentials following current injection (>1 nA, > 5 ms) were discarded from analysis. Membrane potentials were recorded for 1–10 min. Sik3 hypomorph and UAS-Sik3, UAS-Sik3(S563A) transgenic flies were gifts from M. Montminy and J. B. Thomas40. elav-GS (GeneSwitch) stocks were from the Bloomington stock centre. Flies were reared at 25 °C under 12-h light:12-h dark cycle in 50–60% relative humidity on a standard fly food consisting of corn meal, yeast, glucose, wheat germ and agar. Sleep analysis was performed as described previously41. In brief, male flies (2–5 days old) were individually housed in glass tubes (length, 65 mm; inside diameter, 3 mm) containing standard fly food at one end and a cotton plug on the other end. Sucrose-agar (1% agar supplemented with 5% sucrose) food was used for the GeneSwitch system assay, instead of standard food. The glass tubes were placed in the Drosophila activity monitor (DAM) (Trikinetics) and the locomotor activity of each fly was recorded as the number of infrared beam crossings in 1-min bin. Sleep was defined as periods of inactivity lasting 5 min or longer. Sleep assay were performed for 3 d under 12-h light:12-h dark cycle conditions and then constant darkness conditions. For 12-h light:12-h dark cyles, zeitgeber time (ZT) was used, and for constant darkness, circadian time (CT), with CT0 as 12 h after lights-off of the last 12-h light:12-h dark conditions, was used to indicate the daily time. For conditional expression analysis, we used the GeneSwitch system42 where expression is induced by a steroid hormone antagonist RU486. Flies are monitored for 3 days in tubes without drug in constant darkness and then transferred to new tubes either with vehicle (0.5% DMSO) alone or with 0.5 mM RU486 and then further monitored under constant darkness conditions. The expression of endogenous or transgenic Sik3 genes was confirmed by RT–PCR using RNA from fly heads. The wild-type strain N and the mutant strain PY1479 kin-29(oy38) X were obtained from the Caenorhabditis Genetics Center (CGC)43. All worms were maintained at 20 °C on nematode growth medium (NGM) agar plates seeded with E. coli HB101. For construction of P ::kin-29, kin-29 cDNA was amplified by RT–PCR and inserted into the plasmid pPD-DEST (a gift from Y. Iino) to generate pDEST-KIN-29. Next, we carried out the LR-recombinase reaction (Gateway System, Life Technologies) between pENTR-P (a gift from Y. Iino) and pDEST-KIN-29 to generate P ::kin-29. P ::kin-29 was injected at 30 ng μl−1 together with the injection marker P ::mcherry (10 ng μl−1) and the empty vector pPD49_26 (60 ng μl−1) into the kin-29(oy38) mutant worms. Quiescence during the L4 to adult lethargus was measured using the microfluidic-chamber based assay44. In brief, polydimethylsiloxane-made microfluidic chambers containing liquid NGM and the E. coli HB101 were loaded with early L4 larvae and sealed with a cover glass plus 2% agarose, and set under the microscope. Images were taken every 2 s for 12 to 20 h at 20 ± 0.5 °C using the microscope M205FA (Leica) equipped with the camera MC120HD (Leica) (pixel size: 1,024 μm × 768 μm) controlled by Leica Application Suite V4.3 or the microscope SZX16 (Olympus) equipped with the camera GR500BCM2 (Shodensha) (pixel size: 1,024 μm × 768 μm) controlled by μManager (UCSF). Subtraction between serial images was carried out using Image J, and worms were regarded as quiescent at a specific time point if the difference from the preceding time point was less than 1% of the total body size. The fraction of quiescence was defined as the number of quiescent time points divided by the total number of time points during a period of 10 min. The onset of lethargus quiescence was defined as the time point after which the fraction of quiescence was higher than 0.05 for at least 20 min, whereas the end point was defined as the time point after which the fraction of quiescence was lower than 0.05 for at least 20 min. Occasionally, brief episodes of quiescence were observed outside of lethargus both in wild-type and mutant worms; these episodes were excluded by setting a threshold of 60 min for the minimum duration of lethargus quiescence. Sample sizes were determined using R software based on averages and standard deviations that were obtained from small scale experiments. No method of randomization was used in any of the experiments. The experimenters were blinded to genotypes and treatment assignment. Statistical analysis was performed using SPSS Statistics 22 (IBM) and R software. All data were tested for Gaussian distribution and variance. Homogeneity of variances was tested with Levene’s test. We used Student’s t-test for pairwise comparisons, one-way ANOVA for multiple comparisons, one-way repeated measure ANOVA for multiple comparisons with multiple data points, and two-way ANOVA for multiple comparisons involving two independent variables. ANOVA analyses were subjected to Tukey’s post-hoc test. When deviation from normality and lack of homogeneity of variances occurred (P < 0.05), Mann–Whitney U test was used for group comparison. P < 0.05 was considered statistically significant. The datasets generated and/or analysed during the current study are available from the corresponding author on reasonable request.

Agency: Cordis | Branch: FP7 | Program: CP-FP | Phase: HEALTH.2012.2.2.2-1 | Award Amount: 8.22M | Year: 2013

In spite of valuable approaches applied to get a broad understanding of genetic, epidemiologic and molecular and system-level biological principles of human aging, cognitive decline remains as one of the greatest health challenges of the old age, with nearly 50% of adults over 85 afflicted of Alzheimers disease. Furthermore, drug development has not performed as expected in clinical trials, at least in part because of an insufficient mechanistic understanding at the systemic level in human. AgedBrainSYSBIO is a timely and straightforward project based on the integration of available transcriptomics, proteomics and metabolomics data, addition of relevant novel sets of data, their modeling and experimental testing in both human, mouse and drosophila. The concept is to identify subsets of pathways with two unique druggable hallmarks: (i) the validation of interactions occurring locally in subregions of neurons and (ii) a human and/or primate accelerated evolutionary signature, using six interacting approaches: (1) the identification of interacting protein networks from recent Late-Onset Alzheimer Disease- Genome Wide Association Studies (LOAD-GWAS) data, (2) the experimental validation of interconnected networks working in subregion of a neuron (such as dendrites and dendritic spines), (3) the inclusion of these experimentally validated networks in larger networks obtained from available databases to extend possible protein interactions, (4) the identification of human and/or primate positive selection either in coding or in regulatory gene sequences,(5) the manipulation of these human and/or primate accelerated evolutionary interacting proteins in human neurons derived from induced Pluripotent Stem Cells (iPSCs) and modeling prediction challenged in drosophila and novel mouse transgenic models. This work will finally allow (6) the validation of new druggable targets and markers as a proof-of-concept towards the prevention and cure of aging cognitive defects.

Jaschke P.R.,University of British Columbia | Saer R.G.,University of British Columbia | Noll S.,Gene Bridges GmbH | Beatty J.T.,University of British Columbia
Methods in Enzymology | Year: 2011

The α-proteobacterium Rhodobacter sphaeroides is an exemplary model organism for the creation and study of novel protein expression systems, especially membrane protein complexes that harvest light energy to yield electrical energy. Advantages of this organism include a sequenced genome, tools for genetic engineering, a well-characterized metabolism, and a large membrane surface area when grown under hypoxic or anoxic conditions. This chapter provides a framework for the utilization of R. sphaeroides as a model organism for membrane protein expression, highlighting key advantages and shortcomings. Procedures covered in this chapter include the creation of chromosomal gene deletions, disruptions, and replacements, as well as the construction of a synthetic operon using a model promoter to induce expression of modified photosynthetic reaction center proteins for structural and functional analysis. © 2011 Elsevier Inc. All rights reserved.

Vaisman A.,U.S. National Institutes of Health | McDonald J.P.,U.S. National Institutes of Health | Noll S.,Gene Bridges GmbH | Huston D.,U.S. National Institutes of Health | And 3 more authors.
Mutation Research - Fundamental and Molecular Mechanisms of Mutagenesis | Year: 2014

Low fidelity Escherichia coli DNA polymerase V (pol V/UmuD'2C) is best characterized for its ability to perform translesion synthesis (TLS). However, in recA730 lexA(Def) strains, the enzyme is expressed under optimal conditions allowing it to compete with the cell's replicase for access to undamaged chromosomal DNA and leads to a substantial increase in spontaneous mutagenesis. We have recently shown that a Y11A substitution in the "steric gate" residue of UmuC reduces both base and sugar selectivity of pol V, but instead of generating an increased number of spontaneous mutations, strains expressing umuC_Y11A are poorly mutable in vivo. This phenotype is attributed to efficient RNase HII-initiated repair of the misincorporated ribonucleotides that concomitantly removes adjacent misincorporated deoxyribonucleotides. We have utilized the ability of the pol V steric gate mutant to promote incorporation of large numbers of errant ribonucleotides into the E. coli genome to investigate the fundamental mechanisms underlying ribonucleotide excision repair (RER). Here, we demonstrate that RER is normally facilitated by DNA polymerase I (pol I) via classical "nick translation". In vitro, pol I displaces 1-3 nucleotides of the RNA/DNA hybrid and through its 5'→3' (exo/endo) nuclease activity releases ribo- and deoxyribonucleotides from DNA. In vivo, umuC_Y11A-dependent mutagenesis changes significantly in polymerase-deficient, or proofreading-deficient polA strains, indicating a pivotal role for pol I in ribonucleotide excision repair (RER). However, there is also considerable redundancy in the RER pathway in E. coli. Pol I's strand displacement and FLAP-exo/endonuclease activities can be facilitated by alternate enzymes, while the DNA polymerization step can be assumed by high-fidelity pol III. We conclude that RNase HII and pol I normally act to minimize the genomic instability that is generated through errant ribonucleotide incorporation, but that the "nick-translation" activities encoded by the single pol I polypeptide can be undertaken by a variety of back-up enzymes. © 2014.

Mercer R.G.,Memorial University of Newfoundland | Quinlan M.,Memorial University of Newfoundland | Rose A.R.,Memorial University of Newfoundland | Noll S.,Gene Bridges GmbH | And 2 more authors.
FEMS Microbiology Letters | Year: 2012

Production of the gene transfer agent of Rhodobacter capsulatus, RcGTA, is dependent upon several cellular regulatory systems, including a putative phosphorelay involving the CtrA and CckA proteins. These proteins are also involved in flagellar motility in R. capsulatus. The interactions of proteins in this system are best understood in Caulobacter crescentus where CtrA is activated by phosphorylation by the CckA-ChpT phosphorelay. CtrA~P activity is further controlled by SciP, which represses ctrA transcription and CtrA activation of transcription. We show that R. capsulatus chpT and cckA mutants both have greatly reduced motility and RcGTA activity. Unlike the ctrA mutant where RcGTA gene transcription is absent, the decrease in RcGTA activity is because of reduced release of RcGTA from the cells. The sciP mutant is not affected for RcGTA production but our results support the C. crescentus model of SciP repression of flagellar motility genes. We show that both unphosphorylated and phosphorylated CtrA can activate RcGTA gene expression, while CtrA~P seems to be required for release of the particle and expression of motility genes. This has led us to a new model of how this regulatory system controls motility and production of RcGTA in R. capsulatus. © 2012 Federation of European Microbiological Societies.

Maresca M.,TU Dresden | Erler A.,TU Dresden | Fu J.,TU Dresden | Friedrich A.,TU Dresden | And 2 more authors.
BMC Molecular Biology | Year: 2010

Background: The Red proteins of lambda phage mediate probably the simplest and most efficient homologous recombination reactions yet described. However the mechanism of dsDNA recombination remains undefined.Results: Here we show that the Red proteins can act via full length single stranded intermediates to establish single stranded heteroduplexes at the replication fork. We created asymmetrically digestible dsDNA substrates by exploiting the fact that Redα exonuclease activity requires a 5' phosphorylated end, or is blocked by phosphothioates. Using these substrates, we found that the most efficient configuration for dsDNA recombination occurred when the strand that can prime Okazaki-like synthesis contained both homology regions on the same ssDNA molecule. Furthermore, we show that Red recombination requires replication of the target molecule.Conclusions: Hence we propose a new model for dsDNA recombination, termed 'beta' recombination, based on the formation of ssDNA heteroduplexes at the replication fork. Implications of the model were tested using (i) an in situ assay for recombination, which showed that recombination generated mixed wild type and recombinant colonies; and (ii) the predicted asymmetries of the homology arms, which showed that recombination is more sensitive to non-homologies attached to 5' than 3' ends. Whereas beta recombination can generate deletions in target BACs of at least 50 kb at about the same efficiency as small deletions, the converse event of insertion is very sensitive to increasing size. Insertions up to 3 kb are most efficiently achieved using beta recombination, however at greater sizes, an alternative Red-mediated mechanism(s) appears to be equally efficient. These findings define a new intermediate in homologous recombination, which also has practical implications for recombineering with the Red proteins. © 2010 Maresca et al; licensee BioMed Central Ltd.

Gene Bridges Gmbh | Date: 2010-12-17

This invention is related to bacterial engineering and the heterologous expression of useful compounds. In particular, the invention relates to a heterologous host that has been engineered for expression of a gene which is capable of polyketide or non-ribosomal peptide synthesis. Methods of treating cancer are also disclosed.

Genebridges Gmbh | Date: 2013-10-15

The present invention provides a method of preparing electrocompetent cells from Gram-negative bacteria characterized in that the bacteria are prepared under room temperature. Also provided are electrocompetent Gram-negative bacteria and kits which comprise the electrocompetent bacteria.

PubMed | Gene Bridges GmbH and Mount Sinai Medical Center
Type: Journal Article | Journal: BioTechniques | Year: 2015

Huntingtons disease (HD) is a fatal neurodegenerative disorder that is caused by a CAG repeat expansion encoding a polyglutamine tract in the huntingtin (htt) gene. None of the existing HD mouse models recapitulate the exact disease symptoms and course as it is seen in humans and the generation of further HD disease models is challenging because of the size and complexity of the htt gene locus. Starting from a single substrate plasmid harboring human htt cDNA comprising 98 glutamine (Q) residues, we applied Red/ET recombination to generate four BDNF-BAC transgenes harboring full-length or truncated (N171) htt cDNA comprising 98 or 15 Q residues. BDNF (brain-derived neurotrophic factor) is expressed in the cortical neurons projecting to the striatal medium spiny neurons, and was used to direct htt transgene expression to investigate the contribution of these cell types to HD.

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