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17 protocols using b6 cg tg thy1 yfp hjrs j

1

Genetic Mouse Models for Neurodevelopment

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The study protocol was approved by the local animal welfare committee (Comité Local GIN, C2EA-04 – APAFIS number 8303–2016060110523424) and complied with EU guidelines (directive 2010/63/EU). Every precaution was taken to minimize the number of animals used and stress to animals during experiments.
All mouse lines used in this study were on a C57BL6 genetic background. CRMP4-KO (Khazaei et al., 2014 (link)) and their WT littermates were obtained by crossing CRMP4-heterozygous mice. Single and double CRMP4/Sema3E heterozygotes and their WT littermates were obtained by crossing CRMP4-heterozygote with Sema3E-heterozygote mice. Thy1-eYFP line H mice (Feng et al., 2000 (link)) were obtained from Jackson Labs (B6.Cg-Tg(Thy1-YFP) HJrs/J, RRID:IMSR_JAX:003782) and crossed with heterozygous CRMP4 mice to generate the heterozygous Thy1-eYFP CRMP4 mouse colony. These mice were then crossed with heterozygous CRMP4 mice to generate WT/ and KO/Thy1-eYFP-H littermate mice in inbred F1. Male and/or female mice were used from E17.5 prenatal day up to 4 months of age, depending on the experiments.
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2

Organotypic Hippocampal Slice Culture from Thy1-YFP Mice

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Organotypic slice cultures of the hippocampus were prepared from the Thy1-YFP H line mice31 (link). The mice (listed as B6.Cg-Tg (Thy1-YFP) HJrs/J; stock number 003782 in the supplier's catalogue) were purchased from Jackson Laboratory (Maine). The cultures are prepared as previously reported4 (link)32 . Briefly, a newborn pup of either sex was anesthetized and sacrificed at postnatal day 7. The hippocampus from either side was isolated, and its dorsoventrally central 1/3 portion was cut into 250-μm thick slices with a McIlwain tissue chopper. Preliminary examinations indicated that sex and side of the hippocampus did not affect the results (Dr. R. Shigemoto, personal communication). Each slice was laid on a piece of polytetrafluoroethylene filter (~5 × 5 mm), which was placed in an insert of a Millicell CF (Millipore) multiwell dish.
The cultures were maintained in a humidified atmosphere of 34°C for 18 days before beginning experiments. Culture medium, composed of 50% minimal essential medium of Hanks' salt, 25% Hanks' basal salt solution, and 25% heat-inactivated horse serum (all from Gibco), was renewed every 3–4 days.
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3

Transgenic Mouse Model for ALS

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SOD1G37R/YFP (SOD1+/-; YFP+/-) transgenic mice were previously described (Martineau et al., 2018 (link)). Briefly, they were obtained by breeding male loxSOD1G37R mice (RRID:IMSR_JAX:016149) with heterozygote female Thy1-YFP, line H, mice (B6.Cg-Tg(Thy1-YFP)HJrs/J; The Jackson Laboratory, stock number 003782; RRID:IMSR_JAX:003782). Thy1-YFP mice from line H were used due to their sparse expression of the yellow-fluorescent protein (YFP) in lower motor neurons (Feng et al., 2000 (link)) allowing visualization of single motor axons. Parent lines were maintained on a C57BL6/J background. Importantly, transmission of both transgenes follows a Mendelian autosomal pattern of inheritance, ruling out the possibility that either transgene is integrated on a sex chromosome. Disease progression and motor function were monitored via weekly weight and grip strength measurements (BioSeb, BIO-GS3). Animals were sacrificed using a lethal dose of isoflurane. All experiments were performed in accordance with the guidelines of the Canadian Council on Animal Care, the Comité de Déontologie sur l’Expérimentation Animale of Université de Montréal (protocol #18–040), and the CRCHUM Institutional Committee for the Protection of Animals (protocol #N16008CVV and #N15047ADPs).
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4

In Vivo Spine Analysis of α-Synuclein Overexpression

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All experiments involving mice were approved by the University of Iowa Institutional Animal Care and Use Committee (Protocol #9082242 and 2122242). All experiments were performed in strict adherence with the National Institute of Health (NIH) Guide for the Care and Use of Laboratory Animals. Mice were handled until comfortable with experimenter handling and with the Neurotar setup before any imaging sessions commenced.
For in vivo spine analysis studies, we utilized the Thy1-YFP mouse line, encoding the fluorescent molecule, YFP under the control of the Thy1 promoter: B6.Cg-Tg(Thy1-YFP)HJrs/J; stock number 3782 (Jackson Labs), and the Thy1-GFP line for confocal imaging studies: Tg(Thy1-GFP)MJrs/J; stock number 007788 (Jackson Labs). For Thy1-YFP, males and females were used and randomized to either control (n = 6) or α-Synuclein overexpression (n = 8) treatment groups prior to experiment onset. All mice were injected as mature adults, with age range for controls 39–70 weeks old; α-Synuclein overexpression range 40–73 weeks old. The Thy1-GFP mice contained 4 mice (3 m, 1F); age at injection ranged from 11 to 24 weeks old. Mice were housed in groups of 2–4 per cage, except when directed to singly house by the facility veterinarian due to fighting. In general, the cranial window and head bar did not prevent group housing.
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5

Transgenic Mouse Experimental Protocol

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The C57BL/6 mice, FVB/N-Tg(GFAP-eGFP)14Mes/J and B6.Cg-Tg(Thy1-YFP)HJrs/J transgenic mice were all purchased from the Jackson Laboratory (Bar Harbor, ME, USA). The animals were raised in standard housing conditions (22 ± 1 °C; light/dark cycle of 12/12 h), with water and food available ad libitum. All experiments were performed in accordance with the US National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publication No. 8023) and its 1978 revision, and all experimental protocols were approved by the Institutional Animal Care and Use Committee of China Medical University, No. [2019]059.
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6

Transgenic Mouse Model for ALS

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WT/YFP (SOD1-/-;YFP+/-) and SOD1G37R/YFP (SOD1+/-;YFP+/-) double transgenic mice were obtained by crossing transgenic males heterozygote for the human SOD1G37R transgene (flanked by LoxP sites; loxSOD1G37R) to transgenic females heterozygote for the Thy1-YFP transgene (line H; [B6.Cg-Tg(Thy1-YFP)HJrs/J]; The Jackson laboratories, Bar Harbor, ME, stock number 003782) (Feng et al., 2000 (link)). LoxSOD1G37R mice have been described previously (Boillée et al., 2006b (link); Lobsiger et al., 2009 (link)). Single transgenic lines were maintained on a C57BL/6J background. Progenies were genotyped for the SOD1 and the YFP transgenes by PCR performed on a tail sample or an ear punch extract. Mice of both sexes were used. Disease progression was monitored through weekly weighing and all-limb grip strength measurements (BioSeb, FL; BIO-GS3). Animals were sacrificed using a lethal dose of isoflurane. All experiments were performed in accordance with the guidelines of the Canadian Council on Animal Care, the Comité de Déontologie sur l’Expérimentation Animale of Université de Montréal (protocol #18 – 040) and the CRCHUM Institutional Committee for the Protection of Animals (protocol #N16008CVV and #N15047ADPs).
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7

Sarm1 Knockout Mouse Model

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Sarm1 knockout mice (B6.129X1-Sarm1tm1Aidi/J) were obtained from Jackson Laboratory (Bar Harbor, ME) and maintained on a C57BL/6J background. Heterozygous mating produced homozygous Sarm1 knockout (i.e. Sarm1-/-), heterozygotes (i.e. Sarm1+/-) and wild type (i.e. Sarm1+/+) littermates. A colony of Sarm1 knockout mice and wild type littermates expressing neuronal YFP was generated by crossing Sarm1 knockout mice with Thy1-YFP-H transgenic mice (B6.Cg-Tg(Thy1-YFP)HJrs/J, Jackson Laboratory). Sarm1-/-YFP+ mice and Sarm1+/+YFP+ littermates were generated from Sarm1+/-YFP+ x Sarm1+/- mating pairs. Mice were genotyped using the following PCR primers. Sarm1 knockout forward and reverse primers were CTT GGG TGG AGA GGC TAT TC and AGG TGA GAT GAC AGG AGA TC, respectively. Wild type forward and reverse primers were GGG AGA GCC TTC CTC ATA CC and TAA GGA TGA ACA GGG CCA AG, respectively. YFP transgene expression was detected by the presence of neuronal YFP in ear punch samples under fluorescence microscopy. Animals were housed in littermate groups regardless of genotype, on a 12 h light-dark cycle and fed ad libitum. All animal procedures were performed in accordance with the Virginia Commonwealth University Animal Care and Use Program’s regulations under an approved protocol (protocol number: AD10000395).
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8

Generation and Maintenance of Transgenic Mice

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CD63-GFP floxed mice were generated in the lab by homologous recombination, as previously described 35 (link). The WT (C57B/6J, #000664), Ai14-tdTf/f reporter (#007914), ApoE-KO (B6.129P2-Apoe tm1Unc/J #002052), B6.Cg-Tg (Thy1-YFP)HJrs/J (#003782), and B6.C-Tg(CMV-Cre)1Cgn/J(#006054) mice were obtained from the Jackson Laboratory. HepaCAM knock-out (KO) mice were generated by breeding HepaCAM floxed mice (a kind gift from Dr. Cagla Eroglu at Duke University)19 (link) with CMV-Cre mice. Both male and female mice were used in all experiments. All mice were maintained on a 12 h light/dark cycle with food and water ad libitum. Care and treatment of animals in all procedures strictly followed the NIH Guide for the Care and Use of Laboratory Animals and the Guidelines for the Use of Animals in Neuroscience Research. Animal protocols used in this study have been approved by the Tufts University IACUC.
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9

Cyfip2 Conditional Knockout Mouse Model

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The floxed-Cyfip2 and Cyfip2+/− mice used in this study have been previously described (Han et al., 2015 (link); Zhang et al., 2018 (link), 2019a (link); Lee S. H. et al., 2020 (link)). The CaMKIIα-Cre [B6.Cg-Tg(Camk2a-cre)T29-1Stl/J] and Thy1-YFP [B6.Cg-Tg(Thy1-YFP)HJrs/J] mice were obtained from the Jackson Laboratory. The mice were bred and maintained on a C57BL/6J background according to the Korea University College of Medicine Research Requirements. All procedures were approved by the Committee on Animal Research at Korea University College of Medicine (KOREA-2018-0174). The mice were fed ad libitum and housed under a 12 h light-dark cycle. All experiments were performed with an adult (8 to 10-week old) male Cyfip2 mice and their littermate controls.
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10

Thy1-YFPH Mouse Model for Neuronal Tracing

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All experimental procedures involving animals were approved by the Animal Ethics Committee of the University of Tasmania (ethics approval number A0011076) and are in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes. Animals were housed in standard conditions (20°C, 12 h/12 h light/dark cycle) with access to food and water ad libitum and monitored daily for signs of stress and illness. Thy1-YFPH line mice [B6.Cg-Tg(Thy1-YFP)HJrs/J, stock number 003782] were obtained from the Jackson Laboratory (Bar Harbor, ME, United States) and maintained as a heterozygous colony. In these animals YFP is expressed under the control of the neuron-specific Thy1 promoter in ∼80% of layer 5 and 2/3 neocortical pyramidal neurons (Feng et al., 2000 (link)). Ear punches were taken at weaning (4 weeks) to determine inheritance of the YFP transgene. Tissue was mounted on a glass slide and examined with the 488 nm filter on a Leica DM LB2 microscope (Leica Microsystems Pty Ltd., North Ryde, NSW, Australia). Animals carrying the YFP transgene were identified as possessing YFP-positive (YFP+) axons within their ear clips.
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