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Galveston, TX, United States
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News Article | May 23, 2017
Site: www.eurekalert.org

DALLAS - May 22, 2017 - A new DNA vaccine when delivered to the skin prompts an immune response that produces antibodies to protect against toxic proteins associated with Alzheimer's disease - without triggering severe brain swelling that earlier antibody treatments caused in some patients. Two studies from the Peter O'Donnell Jr. Brain Institute demonstrate in animals how a vaccine containing DNA of the toxic beta-amyloid protein elicits a different immune response that may be safe for humans. The vaccine, which will likely be tested further by the U.S. Food and Drug Administration, is on a shortlist of promising antibody treatments that may eventually help settle a high-stakes debate of whether amyloid is a vital target for preventing or curing Alzheimer's. "If you look at the hard reality, the odds are against us because so many therapies have failed through the years. But this has potential," said Dr. Roger Rosenberg, co-author of the studies and Director of the Alzheimer's Disease Center at UT Southwestern Medical Center. Dr. Rosenberg notes that earlier research established that antibodies significantly reduce amyloid buildup in the brain, but he needed to find a safe way to introduce these into the body. A vaccine developed elsewhere showed promise in the early 2000s, but when tested in humans it caused brain swelling in some patients. Dr. Rosenberg's idea was to start with DNA coding for amyloid and inject it into the skin rather than the muscle. The injected skin cells make the amyloid protein, and the body responds by producing new antibodies that inhibit the buildup of amyloid, which some scientists blame for destroying neurons. Although the DNA vaccine has not yet been tested in humans, it produces a different kind of immune response in the tested animals that significantly lessens the chance of an adverse response in the brain, according to the studies published in the Journal of Alzheimer's Disease and Alzheimer's Research & Therapy. The research is notable because it shows a DNA vaccine can be effective and safe in two large mammals. Most other vaccines only produced an immune response in mice but not large mammals. "We believe this kind of immune response has a high probability of being safe in humans and also being effective to make high levels of antibody," said Dr. Rosenberg, Professor of Physiology, Neurology and Neurotherapeutics. Alzheimer's disease is characterized by progressive deterioration of the brain as neurons are destroyed. More than 5 million Americans have the fatal disease, with the number expected to nearly triple by 2050, according to the Centers for Disease Control and Prevention. No known cure exists, though an array of antibody and other treatments are being researched to target amyloid plaques. One strategy involves preforming the antibodies in the laboratory and inserting them into the body - a technique that is still being tested for clinical benefits. Dr. Rosenberg said there would be distinct advantages to allowing the body to produce its own antibodies through active immunization, if it can be done safely. Among them, the vaccine would be more accessible and less expensive. It also produces a wider variety of antibody types than the preformed antibodies, he said. "All the vaccines we received as kids and adults have been active vaccinations; we made the antibodies in the body," Dr. Rosenberg said. "It's safer, more effective, and it's sustained longer." Dr. Rosenberg's research is the latest contribution to decades of study across the globe focusing on clearing amyloid plaques in hopes of curing or slowing the progression of Alzheimer's. A lack of results over the years has prompted some scientists to question whether they are properly targeting the disease. A British study from 2008 showed that removing amyloid after it accumulates in the brain does not improve brain cognition. The findings highlight a couple of lingering questions that have crucial implications for the future of Alzheimer's research: Is amyloid merely a symptom, not the cause of the disease? And if there is causation, can earlier treatments make a difference? Dr. Rosenberg acknowledges that preventing amyloid buildup by itself may not be an adequate treatment for Alzheimer's, but it could be a major part of the solution. He and other researchers at UT Southwestern are also studying the potential benefits of preventing and removing tangles of toxic tau proteins from the brain, another hallmark of the disease. "Some in the scientific community believe the reason amyloid therapies have failed so far is because too little of the therapy was given, and too late," Dr. Rosenberg said. "The jury is still out." Dr. Rosenberg's latest studies show the potential of a DNA vaccine to prevent the buildup of amyloid in otherwise healthy people. The vaccine was administered to healthy animals, inducing an anti-inflammatory immune response of up to 40 times more anti-amyloid antibodies than an earlier vaccine Dr. Rosenberg tested a decade ago. Dr. Rosenberg expects the FDA will want further tests of the vaccine in its own labs before planning a potential clinical trial on people. If proven effective, the vaccine could be given to people who are at risk of developing Alzheimer's but have not yet started forming amyloid plaques. Dr. Rosenberg keeps his expectations in check, noting the billions of dollars and multitude of studies that have so far yielded little advancement in treating Alzheimer's disease. "Finding answers to this disease will knock you down fast," said Dr. Rosenberg, who has worked at UT Southwestern for 44 years and holds the Abe (Brunky), Morris and William Zale Distinguished Chair in Neurology. "I've made a commitment to this place and to this research. I'm trying, and I'll keep going." Study collaborators include co-author Dr. Doris Lambracht-Washington, Instructor of Neurology and Neurotherapeutics, and Min Fu, Senior Research Associate. The Texas Biomedical Research Institute in San Antonio and Animal Resource Center in Dallas helped in the studies. The research was funded by the National Institute on Aging, the Zale Foundation, the McCune Foundation, the Rudman Foundation, the Alliance of Women for Alzheimer's Research and Education, Presbyterian Village North Foundation; and Freiberger, Losinger, and Denker Family Funds. UT Southwestern, one of the premier academic medical centers in the nation, integrates pioneering biomedical research with exceptional clinical care and education. The institution's faculty has received six Nobel Prizes, and includes 22 members of the National Academy of Sciences, 18 members of the National Academy of Medicine, and 14 Howard Hughes Medical Institute Investigators. The faculty of more than 2,700 is responsible for groundbreaking medical advances and is committed to translating science-driven research quickly to new clinical treatments. UT Southwestern physicians provide care in about 80 specialties to more than 100,000 hospitalized patients, 600,000 emergency room cases, and oversee approximately 2.2 million outpatient visits a year. This news release is available on our website at http://www. To automatically receive news releases from UT Southwestern via email, subscribe at http://www.


Lambracht-Washington D.,outhwestern Medical Center Dallas | Fu M.,outhwestern Medical Center Dallas | Wight-Carter M.,Animal Resource Center | Riegel M.,Animal Resource Center | And 2 more authors.
Journal of Alzheimer's Disease | Year: 2017

A pathological hallmark of Alzheimer's disease (AD) are amyloid plaques in the brain consisting of aggregated amyloid-β 42 peptide (Aβ42) derived from cellular amyloid-β protein precursor (AβPP). Based on successful experiments in mouse AD models, active immunization with Aβ42 peptide and passive immunizations with anti-Aβ42 antibodies were started in clinical trials. Active Aβ42 peptide immunization in humans had led to an inflammatory autoimmune response, and the trial was stopped. Passive immunizations had shown some effects in slowing AD pathology. Active DNA Aβ42 immunizations administered with the gene gun into the skin elicits a different immune response and is non-inflammatory. While in rodents, good responses had been found for this type of immunization, positive results in larger mammals are missing. We present here results from sixteen New Zealand White Rabbits, which underwent intradermal DNA Aβ42 immunization via gene gun. The humoral immune response was analyzed from blood throughout the study, and cellular immune responses were determined from spleens at the end of the study. A good anti-Aβ antibody response was found in the rabbit model. The T cell response after re-stimulation in cell culture showed no IFNγ producing cells when ELISPOT assays were analyzed from PBMC, but low numbers of IFNγ and IL-17 producing cells were found in ELISPOTS from spleens (both 5 immunizations). Brains from immunized rabbits showed no signs of encephalitis. Based on these results, DNA Aβ42 immunization is highly likely to be safe and effective to test in a possible clinical AD prevention trial in patients. © 2017 - IOS Press and the authors. All rights reserved.


Zhang X.,U.S. Food and Drug Administration | Newport G.D.,U.S. Food and Drug Administration | Callicott R.,Animal Resource Center | Liu S.,U.S. Food and Drug Administration | And 8 more authors.
Neurotoxicology and Teratology | Year: 2016

Methylphenidate (MPH) is a psychostimulant commonly used for the treatment of Attention-Deficit Hyperactivity Disorder (ADHD). Since the long-term effects of this drug on the central nervous system (CNS) are not well understood, we conducted microPET/CT scans on young adult male rhesus monkeys (n = 4/group) to gather information on brain metabolism using the uptake of [18F]Fluoro-2-deoxy-2-D-glucose (FDG) as a marker. Approximately two-year old, male rhesus monkeys were treated orally with MPH twice per day, five days per week (M-F) over a 6-year period. Subjects received MPH at either 2.5 or 12.5 mg/kg/dose or vehicle (Prang). To minimize the acute effects of MPH on FDG uptake, microPET/CT scans were scheduled on Mondays before their first daily dosing of the week (approximately 68 h since their last treatment). FDG (370 ± 8.88 MBq) was injected intravenously and 30 min later microPET/CT images were obtained over 60 min. Radiolabeled tracer accumulation in regions of interest (ROIs) in the prefrontal cortex, temporal cortex, striatum and cerebellum were converted into Standard Uptake Values (SUVs). Compared to the control group, the uptake of FDG in the cerebellum was significantly decreased in both the low and high dose groups. These preliminary data demonstrate that microPET imaging is capable of distinguishing differences in retention of FDG in the brains of NHPs treated chronically with MPH and suggests that this approach may provide a minimally invasive biomarker for exploring the effects of chronic MPH treatment on aspects of brain function. © 2016


Lim R.,Monash Institute of Medical Research | Zavou M.J.,Monash Institute of Medical Research | Milton P.-L.,Animal Resource Center | Chan S.T.,Monash Institute of Medical Research | And 5 more authors.
Journal of visualized experiments : JoVE | Year: 2014

Respiratory dysfunction is one of the leading causes of morbidity and mortality in the world and the rates of mortality continue to rise. Quantitative assessment of lung function in rodent models is an important tool in the development of future therapies. Commonly used techniques for assessing respiratory function including invasive plethysmography and forced oscillation. While these techniques provide valuable information, data collection can be fraught with artefacts and experimental variability due to the need for anesthesia and/or invasive instrumentation of the animal. In contrast, unrestrained whole-body plethysmography (UWBP) offers a precise, non-invasive, quantitative way by which to analyze respiratory parameters. This technique avoids the use of anesthesia and restraints, which is common to traditional plethysmography techniques. This video will demonstrate the UWBP procedure including the equipment set up, calibration and lung function recording. It will explain how to analyze the collected data, as well as identify experimental outliers and artefacts that results from animal movement. The respiratory parameters obtained using this technique include tidal volume, minute volume, inspiratory duty cycle, inspiratory flow rate and the ratio of inspiration time to expiration time. UWBP does not rely on specialized skills and is inexpensive to perform. A key feature of UWBP, and most appealing to potential users, is the ability to perform repeated measures of lung function on the same animal.


PubMed | Animal Resource Center and St Jude Childrens Research Hospital
Type: Journal Article | Journal: The Journal of infectious diseases | Year: 2015

An effective vaccine is urgently needed against the H7N9 avian influenza virus. We evaluated the immunogenicity and protective efficacy of a split-virion H7N9 vaccine with or without the oil-in-water adjuvants in ferrets.Ferrets were vaccinated with 2 doses of unadjuvanted, MF59 or AS03-adjuvanted A/Shanghai/2/2013 (H7N9) vaccine, and the induction of antibodies to hemagglutinin (HA) or neuraminidase proteins was evaluated. Ferrets were then challenged with wild-type H7N9 virus to assess the vaccines protective efficacy. The vaccine composition and integrity was also evaluated in vitro.Adjuvanted vaccines stimulated robust serum antibody titers against HA and neuraminidase compared with the unadjuvanted vaccines. Although there was a difference in adjuvanticity between AS03 and MF59 at a lower dose (3.75 g of HA), both adjuvants induced comparable antibody responses after 2 doses of 15 g. On challenge, ferrets that received adjuvanted vaccines showed lower viral burden than the control or unadjuvanted vaccine group. In vitro examinations revealed that the vaccine contained visible split-virus particles and retained the native conformation of HA recognizable by polyclonal and monoclonal antibodies.The adjuvanted H7N9 vaccines demonstrated superior immunogenicity and protective efficacy against H7N9 infection in ferrets and hold potential as a vaccination regimen.


PubMed | Animal Resource Center, Wake Forest Institute for Regenerative Medicine and Monash Institute of Medical Research
Type: | Journal: Journal of visualized experiments : JoVE | Year: 2014

Respiratory dysfunction is one of the leading causes of morbidity and mortality in the world and the rates of mortality continue to rise. Quantitative assessment of lung function in rodent models is an important tool in the development of future therapies. Commonly used techniques for assessing respiratory function including invasive plethysmography and forced oscillation. While these techniques provide valuable information, data collection can be fraught with artefacts and experimental variability due to the need for anesthesia and/or invasive instrumentation of the animal. In contrast, unrestrained whole-body plethysmography (UWBP) offers a precise, non-invasive, quantitative way by which to analyze respiratory parameters. This technique avoids the use of anesthesia and restraints, which is common to traditional plethysmography techniques. This video will demonstrate the UWBP procedure including the equipment set up, calibration and lung function recording. It will explain how to analyze the collected data, as well as identify experimental outliers and artefacts that results from animal movement. The respiratory parameters obtained using this technique include tidal volume, minute volume, inspiratory duty cycle, inspiratory flow rate and the ratio of inspiration time to expiration time. UWBP does not rely on specialized skills and is inexpensive to perform. A key feature of UWBP, and most appealing to potential users, is the ability to perform repeated measures of lung function on the same animal.


PubMed | U.S. Food and Drug Administration, Animal Resource Center and University of Arkansas for Medical Sciences
Type: | Journal: Neurotoxicology and teratology | Year: 2016

Methylphenidate (MPH) is a psychostimulant commonly used for the treatment of Attention-Deficit Hyperactivity Disorder (ADHD). Since the long-term effects of this drug on the central nervous system (CNS) are not well understood, we conducted microPET/CT scans on young adult male rhesus monkeys (n=4/group) to gather information on brain metabolism using the uptake of [(18)F]Fluoro-2-deoxy-2-d-glucose (FDG) as a marker. Approximately two-year old, male rhesus monkeys were treated orally with MPH twice per day, five days per week (M-F) over a 6-year period. Subjects received MPH at either 2.5 or 12.5mg/kg/dose or vehicle (Prang). To minimize the acute effects of MPH on FDG uptake, microPET/CT scans were scheduled on Mondays before their first daily dosing of the week (approximately 68h since their last treatment). FDG (3708.88MBq) was injected intravenously and 30min later microPET/CT images were obtained over 60min. Radiolabeled tracer accumulation in regions of interest (ROIs) in the prefrontal cortex, temporal cortex, striatum and cerebellum were converted into Standard Uptake Values (SUVs). Compared to the control group, the uptake of FDG in the cerebellum was significantly decreased in both the low and high dose groups. These preliminary data demonstrate that microPET imaging is capable of distinguishing differences in retention of FDG in the brains of NHPs treated chronically with MPH and suggests that this approach may provide a minimally invasive biomarker for exploring the effects of chronic MPH treatment on aspects of brain function.


News Article | November 25, 2015
Site: www.nature.com

All mice were maintained on a C57BL/6 background, including ScfGFP (ref. 19), Scffl/+ (ref. 19), Cxcl12DsRed (ref. 18), Cxcl12fl/+ (ref. 18), R26tdTomato (ref. 26), Vav1-cre (ref. 24), Leprcre (ref. 27), Tcf21cre/ER (ref. 21) and α-catulinGFP. To induce Cre/ER activity in Tcf21cre/ER mice, 4–6-week-old mice were administered 2 mg tamoxifen (Sigma) daily by oral gavage for 12 consecutive days. For induction of EMH, mice were injected at day 0 with a single dose of 4 mg cyclophosphamide followed by daily injections of 5 μg G-CSF for 4–21 days. Both male and female mice were used. All mice were housed in the Animal Resource Center at the University of Texas Southwestern Medical Center (UTSW). All procedures were approved by the UTSW Institutional Animal Care and Use Committee. Bone marrow cells were isolated by flushing the femur or tibia with Ca2+- and Mg2+-free HBSS with 2% heat-inactivated bovine serum using a 3 ml syringe fitted with a 25-gauge needle. Spleen cells were obtained by crushing the spleen between two frosted slides. The cells were dissociated to a single-cell suspension by gently passing through the needle several times and then filtering through a 40-μm nylon mesh. Blood was collected by cardiac puncture, and white blood cells were isolated by ficoll centrifugation according to the manufacturer’s instructions (GE Healthcare). The following antibodies were used to isolate HSCs: anti-CD150 (TC15-12F12.2), anti-CD48 (HM48-1), anti-Sca-1 (E13-161.7), anti-c-kit (2B8) and the following antibodies against lineage markers (anti-Ter119, anti-B220 (6B2), anti-Gr-1 (8C5), anti-CD2 (RM2-5), anti-CD3 (17A2), anti-CD5 (53-7.3) and anti-CD8 (53-6.7)). Haematopoietic progenitors were identified by flow cytometry using the following antibodies: anti-Sca-1 (E13-161.7), anti-c-Kit (2B8) and the following antibodies against lineage markers (anti-Ter119, anti-B220 (6B2), anti-Gr-1 (8C5), anti-CD2 (RM2-5), anti-CD3 (17A2), anti-CD5 (53-7.3) and anti-CD8 (53-6.7)), anti-CD34 (RAM34), anti-CD135 (Flt3) (A2F10), anti-CD16/32 (FcγR) (93), anti-CD127 (IL7Rα) (A7R34), anti-CD24 (M1/69), anti-CD43 (1B11), anti-B220 (6B2), anti-IgM (II/41), anti-CD3 (17A2), anti-Gr-1 (8C5), anti-Mac-1 (M1/70), anti-CD41 (MWReg30), anti-CD71 (C2) and anti-Ter119. 4′,6-Diamidino-2-phenylindole (DAPI) was used to exclude dead cells. Antibodies were obtained from eBioscience or BD Bioscience. To isolate bone marrow stromal cells the marrow was gently flushed out of the bone marrow cavity with a 3-ml syringe fitted with a 23-guage needle and then transferred into 1 ml pre-warmed bone marrow digestion solution (200 U ml−1 DNase I (Sigma), 250 μg ml−1 LiberaseDL (Roche) in HBSS plus Ca2+ and Mg2+) and incubated at 37 °C for 30 min with gentle shaking. To isolate splenic stromal cells, the spleen capsule was cut into ~1 mm3 fragments using scissors and then digested as described earlier in spleen digestion solution (200 U ml−1 DNase I, 250 μg ml−1 LiberaseDL, 1 mg ml−1 collagenase, type 4 (Roche) and 500 μg ml−1 collagenase D (Roche) in HBSS plus Ca2+ and Mg2+). After a brief vortex, the spleen fragments were allowed to sediment for ~3 min and the supernatant was transferred to another tube on ice. The sedimented (undigested) spleen fragments were subjected to a second round of digestion. The two fractions of digested cells were pooled and filtered through a 100-μm nylon mesh. Anti-PDGFR-α (APA5), anti-PDGFR-β (APB5), anti-LepR (R&D), anti-CD45 (30F-11) and anti-Ter119 antibodies were used to isolate stromal cells. For analysis of endothelial cells, mice were injected intravenously into the retro-orbital venous sinus with 10 μg Alexa-Fluor-660-conjugated anti-VE-cadherin antibody (BV13) 10 min before being killed. Samples were analysed using a FACSAria or FACSCanto II flow cytometer (BD Biosciences). To assess BrdU incorporation into spleen cells after EMH induction, mice were intraperitoneally injected with a single dose of BrdU (2 mg BrdU per mouse) then maintained on 0.5 mg BrdU per ml drinking water for 7 days. Endothelial cells were labelled by intravenous injection of an anti-VE-cadherin antibody (eBioscience). Enzymatically dissociated spleen cells were stained with antibodies against surface markers and the target cell populations were sorted then resorted to ensure purity. The sorted cells were then fixed, and stained with an anti-BrdU antibody using the BrdU APC Flow Kit (BD Biosciences) according to the manufacturer’s instructions. Adult recipient mice were irradiated using an XRAD 320 X-ray irradiator (Precision X-Ray) with two doses of 540 rad (total 1,080 rad) delivered at least 2 h apart. Cells were injected into the retro-orbital venous sinus of anaesthetized mice. Sorted doses of splenocytes from donor mice with EMH were transplanted along with 3 × 105 recipient bone marrow cells. Recipient mice were bled every 4 weeks to assess the level of donor-derived blood cells, including myeloid, B and T cells for at least 16 weeks. Blood was subjected to ammonium chloride/potassium red cell lysis before antibody staining. Antibodies including anti-CD45.2 (104), anti-CD45.1 (A20), anti-Gr1 (8C5), anti-Mac-1 (M1/70), anti-B220 (6B2) and anti-CD3 (KT31.1) were used for flow cytometric analysis. For bone marrow sections, freshly dissected bones were fixed in 4% paraformaldehyde overnight followed by 3 days of decalcification in 10% EDTA dissolved in PBS. Bones were sectioned using the CryoJane tape-transfer system (Instrumedics). For spleen sections, freshly dissected spleens were fixed in 4% paraformaldehyde for 1 h followed by 1 day incubation in 10% sucrose in PBS. Frozen spleens were sectioned with a cryostat (Leica). For whole mount imaging, spleens were sectioned into ~2 mm pieces. Spleen sections were blocked in PBS with 10% horse serum for 1 h and then stained overnight with chicken-anti-GFP (Aves) and/or rabbit-anti-laminin (Abcam) antibodies. Donkey-anti-chicken Alexa Fluor 488 and/or donkey-anti-rabbit Alexa Fluor 647 were used as secondary antibodies (Invitrogen). Specimens were mounted with anti-fade prolong gold (Invitrogen) and images were acquired with either a Zeiss LSM780 confocal microscope or a Leica SP8 confocal microscope equipped with a resonant scanner. Three-dimensional images were achieved using Bitplane Imaris v.7.7.1 software. Spleens were harvested and fixed for 4 h in 4% PFA at 4 °C. Since the spleen capsule is highly autofluorescent, spleens were sectioned perpendicular to the long axis into 300-μm-thick sections using a Leica VT100S vibrotome. These 300-μm sections were fixed for an additional 2 h in 4% PFA and blocked overnight in staining solution (10% dimethylsulfoxide (DMSO), 0.5% IgePal630 (Sigma) and 5% donkey serum (Jackson Immunoresearch) in PBS). All staining steps were performed in staining solution on a rotator at room temperature. Spleen sections were stained for 3 days in primary antibodies, washed overnight in several changes of PBS then stained for 3 days in secondary antibodies. The stained sections were dehydrated in a methanol dehydration series then incubated for 3 h in 100% methanol with several changes. The methanol was then exchanged with benzyl alcohol:benzyl benzoate 1:2 mix (BABB clearing28). The tissues were incubated in BABB for 3 h to overnight with several exchanges of fresh BABB. Spleen sections were mounted in BABB between two coverslips and sealed with silicone (Premium waterproof silicone II clear; General Electric). We found it necessary to clean the BABB of peroxides (which can accumulate as a result of exposure to air and light) by adding 10 g of activated aluminium oxide (Sigma) to 40 ml of BABB and rotating for at least 1 h, then centrifuging at 2,000 g for 10 min to remove the suspended aluminium oxide particles. Images were acquired using a Zeiss LSM780 confocal microscope with a Zeiss LD LCI Plan-Apo ×25/0.8 multi-immersion objective lens, which has a 570 μm working distance. Images were taken at 512 × 512 pixel resolution with 2 μm Z-steps, pinhole for the internal detector at 47.7 μm. Random spots were inserted into images by generating randomized X, Y, and Z coordinates using the random integer generator at http:// www.random.org. After mouse anaesthesia by ketamine/xylazine, a ventral midline incision was made and the peritoneum was breached. The splenic blood vessels were ligated with an absorbable suture (4-0 vicryl). The splenic vessels were cut distal to the suture and the spleen was removed. The vessels were cauterized and the abdomen was sutured with non-absorbable sutures (3-0 Tevdek III). Buprenorphine was administered every 12 h for 3 days to minimize postoperative pain and mice were maintained with ampicillin-containing water to avoid infection. Complete blood counts were measured one month after the survival surgery. EMH was induced by repeated bleeding over a 2-week period according to a published protocol2. Briefly, 4–6 month-old mice were bled via the tail vein five times, every 3 days, removing approximately 250 μl of blood each time, then the mice were killed for analysis 2 days after the last bleed. Approximately 30,000 CD45−Ter119−VE-cadherin+ splenic endothelial cells were flow cytometrically sorted into 50 μl of 66% trichoracetic acid (TCA) in water. Extracts were incubated on ice for at least 15 min and centrifuged at 16,100 g at 4 °C for 10 min. Precipitates were washed in acetone twice and the dried pellets were solubilized in 9 M urea, 2% Triton X-100, and 1% dithiothreitol (DTT). Samples were separated on 4–12% Bis-Tris polyacrylamide gels (Invitrogen) and transferred to PVDF membrane (Millipore). The blots were incubated with primary antibodies overnight at 4 °C and then with secondary antibodies. Blots were developed with the SuperSignal West Femtochemiluminescence kit (Thermo Scientific). Primary antibodies used: rabbit-anti-SCF (Abcam, 1:1,000) and mouse-anti-actin (Santa Cruz, clone AC-15, 1:20,000). Cells were sorted directly into Trizol (Life Technologies). Total RNA was extracted according to the manufacturer’s instructions. Total RNA was reverse transcribed using SuperScript III Reverse Transcriptase (Life Technologies). Quantitative real-time PCR was performed using SYBR green on a LightCycler 480 (Roche). β-Actin was used to normalize the RNA content of samples. Primers used in this study were Scf: 5′-GCCAGAAACTAGATCCTTTACTCCTGA-3′ and 5′-CATAAATGGTTTTGTGACACTGACTCTG-3′; β-actin: 5′-GCTCTTTTCCAGCCTTCCTT-3′ and 5′-CTTCTGCATCCTGTCAGCAA-3′. Three independent samples of 5,000 spleen Scf-GFP+VE-cadherin− spleen stromal cells and two independent samples of 5,000 unfractionated spleen cells were flow cytometrically sorted into Trizol. Total RNA was extracted, amplified, and sense strand cDNA was generated using the Ovation Pico WTA System V2 (NuGEN) according to the manufacturer’s instructions. cDNA was fragmented and biotinylated using the Encore Biotin Module (NuGEN) according to the manufacturer’s instructions. Labelled cDNA was hybridized to Affymetrix Mouse Gene ST 1.0 chips according to the manufacturer’s instructions. Expression values for all probes were normalized and determined using the robust multi-array average (RMA) method29. Panels in all figures represented multiple independent experiments performed on different days with different mice. Sample sizes were not based on power calculations. No randomization or blinding was performed. No animals were excluded from analysis. Variation is always indicated using standard deviation. For analysis of the statistical significance of differences between two groups we generally performed two-tailed Student’s t-tests. For analysis of the statistical significance of differences among more than two groups, we performed repeated measures one-way analysis of variance (ANOVA) tests with Greenhouse–Geisser correction (variances between groups were not equal) and Tukey’s multiple comparison tests with individual variances computed for each comparison. To assess the statistical significance of differences in fetal mass between paired control and mutant mice (Fig. 5j and Extended Data Fig. 8v), we performed a two-way ANOVA.


Hernandez C.M.,University of Texas Medical Branch | Cortez I.,University of Texas Medical Branch | Gu Z.,U.S. National Institutes of Health | Colon-Saez J.O.,U.S. National Institutes of Health | And 6 more authors.
Journal of Physiology | Year: 2014

There is much interest in α7 nicotinic acetylcholine receptors (nAChRs) in CNS function since they are found throughout peripheral tissues as well as being highly expressed in brain regions implicated in attention, learning and memory. As such, the role of these receptors in many aspects of CNS function and disease is being actively investigated. To date, only one null mouse model (A7KO) is available which is non-conditional and constitutive. Since α7 nAChRs are present on neurons and glia (including astrocytes), as well as being developmentally regulated, there is an unmet need for the technical capability to control α7 nAChR gene expression. Therefore we have generated mice in which the fourth exon of the α7 nAChR gene (Chrna7) is flanked by loxP sites (B6-Chrna7LBDEx4007Ehs) which we refer to as floxed α7 nAChR conditional knockout or α7nAChRflox. We validated the chosen approach by mating α7nAChRflox with mice expressing Cre recombinase driven by the glial acidic fibrillary protein (GFAP)-Cre promoter (GFAP-A7KO) to test whether α7nAChRflox, GFAP-A7KO and appropriate littermate controls performed equally in our standard Rodent In Vivo Assessment Core battery to assess general health, locomotion, emotional and cognitive behaviours. Neither α7nAChRflox nor GFAP-A7KO exhibited significant differences from littermate controls in any of the baseline behavioural assessments we conducted, similar to the 'first generation' non-conditional A7KO mice. We also determined that α7 nAChR binding sites were absent on GFAP-positive astrocytes in hippocampal slices obtained from GFAP-A7KO offspring from α7nAChRflox and GFAP-Cre crosses. Finally, we validated that Cre recombinase (Cre)-mediated excision led to functional, cell- and tissue-specific loss of α7 nAChRs by demonstrating that choline-induced α7 nAChR currents were present in Cre-negative, but not synapsin promoter-driven Cre-positive, CA1 pyramidal neurons. Additionally, electrophysiological characterization of α7 nAChR-mediated current traces was similar in terms of amplitude and time constants of decay (during desensitization) for the α7nAChRflox and wild-type (WT) mice. Thus, we have in vivo and in vitro evidence that the Chrna7 exon 4 targeting strategy does not alter behavioural, cognitive, or electrophysiological properties compared to WT and that Cre-mediated excision is an effective approach to delete α7 nAChR expression in a cell-specific manner. © 2014 The Physiological Society.

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