A PL253 targeting vector containing the Mlh3-D1185N point mutation in the potential endonuclease domain and a loxP-neo-loxP cassette in intron 5–6 of Mlh3 was incorporated into an embryonic stem cell line. Mlh3DN transgenic mice were crossed with a Spo11-Cre mouse line to remove the neo cassette [76 (link)], and then maintained on an inbred background through backcrossing on to the C57Bl/6J line (Jackson Laboratory, Bar Harbor, ME). Genotyping of WT, Mlh3+/DN, and Mlh3DN/DN mice was performed using the following PCR primer pairs: forward (5’-AAGCCAAGTCTGCATGAGTA-3’) and reverse (5’-TAAATGTGCCACTGACTAAAT-3’) followed by a restriction enzyme digestion with Sau96I (New England Biolabs) at 37°C for 2–3 hours, which results in 439-bp and 263-bp fragments from the WT allele and a 702-bp fragment from the mutant allele. Fertility tests were performed by breeding Mlh3DN/DN adult males with WT females. At least 3 males of each genotyped were evaluated. Presence of a copulation plug the following morning counted as a successful mating event. Pregnancy was confirmed by gentle palpation of the abdomen after gestation day 11 or on delivery date of litters. Mus81-/- animals were generated from our breeding stock of such mice, as previously described [6 (link)]. Mice were housed and utilized under the guidance and approval of the Cornell University Institutional Animal Care and Use Committee.
C57bl 6j line
Manufactured by Jackson ImmunoResearch
Sourced in Montenegro
C57BL/6J is a well-characterized inbred mouse strain. It is a widely used model for various research applications.
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7 protocols using c57bl 6j line
1
Gene Targeting to Generate Mlh3-D1185N Mice
2
Age-related Changes in C57BL/6J Mice
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Male C57BL/6J mice of 3 weeks (3 W), 6, 18, and 24 months (6 M, 18 M, and 24 M, respectively) were used in this study. All mice used in this study were the C57BL/6J line originally from Jackson Labs (Bar Harbor, ME). Animals (18 M, 24 M) were supplied by Prof. A Qin (Shanghai Ninth People’s Hospital) and young animals (3 W and 6 M) were purchased from the Shanghai Jihui Laboratory Animal Care Co., Ltd (Shanghai, China). All experimental procedures were approved by the ethical committee of Shanghai Ninth People’s Hospital (No. SH9H-2020-A420-1) and conducted at the Shanghai Institute of Precision Medicine and Ear Institute of Shanghai Jiao Tong University School of Medicine.
3
PV-Cre Transgenic Mice Protocol
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All mice were male parvalbumin-Cre (PV-Cre) heterozygotes, derived from PV-Cre BAC
transgenics back-crossed into a C57BL/6J line (Jackson Laboratory strain B6;
129P2-Pvalbtm1(cre)Arbr). Mice were 8–12 weeks old at the start of training
(mean age = 10.8 ± 1.5 weeks) and 10–15 weeks old at the time of
surgery (mean age = 13.5 ± 2.1 weeks). Animals were individually housed and
maintained on a 12-hr light/dark cycle (lights on at 7:00 AM). All experimental
procedures and animal care protocols were approved by the Massachusetts Institute of
Technology Institutional Animal Care and Use Committees and were in accordance with
NIH guidelines.
transgenics back-crossed into a C57BL/6J line (Jackson Laboratory strain B6;
129P2-Pvalbtm1(cre)Arbr). Mice were 8–12 weeks old at the start of training
(mean age = 10.8 ± 1.5 weeks) and 10–15 weeks old at the time of
surgery (mean age = 13.5 ± 2.1 weeks). Animals were individually housed and
maintained on a 12-hr light/dark cycle (lights on at 7:00 AM). All experimental
procedures and animal care protocols were approved by the Massachusetts Institute of
Technology Institutional Animal Care and Use Committees and were in accordance with
NIH guidelines.
4
Generation and Characterization of miR-146a Mice
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Transgenic founder animals from each line were individually backcrossed three times to the C57BL6/J line (Jackson) and breeding colonies were separately maintained for each line, using heterozygotes for each mutant allele to produce paired homozygous mutant and wild-type control animals for each line. Age- and sex-matched mice from each of the two lines were used for all experiments (except where the use of sex-matched littermates is explicitly noted) such that homozygous mutants from each line were compared with an equivalent number of wild-type mice equally derived from the same two lines. No differences were noted in the phenotypic comparison of wild-type mice derived from breeding of mir-146a+/− heterozygotes and wild-type mice derived from breeding of mir-146a3′F/− heterozygotes in any experiments. All mouse procedures were approved by the Institutional Animal Care and Use Committees of the Baylor College of Medicine.
5
Generation of Neural Progenitor Knockout Mice
6
Generation of Neural Progenitor Knockout Mice
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Experiments were performed in accordance with the guidelines for animal care of the Institutional Animal Care and Use Committee of the University of North Carolina at Chapel Hill under animal protocol 19–209. All mouse (Mus musculus) lines were maintained in the C57BL/6J background by regular backcrossing to the C57BL/6J line (stock number 000664; The Jackson Laboratory). Male and female mice from E15-PND120 were used in experiments, unless otherwise indicated. To generate neural progenitor-specific βII-spectrin null (Sptbn1flox/flox/Nestin-Cre, βIISp-KO) and haploinsufficient (Sptbn1flox/+/Nestin-Cre, βIISp-Het) mice, Sptbn1flox/flox animals (a gift from Mathew Rasband52 (link)) were crossed with the Nestin-Cre mouse line (B6.Cg-Tg(Nes-cre)1Kln/J, stock number 003771; The Jackson Laboratory). Sptbn1flox/flox animals negative for the Cre transgene were used as littermate controls in all experiments. Mice lacking βII-spectrin in cortical projection neurons (Sptbn1flox/flox/Nex-Cre, βIISp-NexKO) were generated by crossing Sptbn1flox/flox and Nex-Cre (a gift from Klaus-Armin Nave55 (link)) animals for multiple generations. All mice were housed at 22 °C ± 2 °C on a 12-hlight/12-h-dark cycle and fed ad libitum regular chow and water.
7
Transgenic Mouse Models for Neural Research
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Mice from the C57BL/6J line (also referred to as wild-type, WT), the CaV3.3 KO line and the VGAT-Ires-Cre line (Jackson Labs, generated by Dr. B. Lowell, Beth Israel Deaconess Medical Center, Harvard) (Vong et al., 2011 (link)) were bred on a C57BL/6J background and housed in a temperature- and humidity-controlled animal house with a 12 hr/12 hr light-dark cycle (lights on at 9 am). Food and water were available ad libitum. For viral injections, 3- to 4-week-old mice of either sex were transferred to a P2 safety level housing room with identical conditions 1 day prior to injection. Then, for in vitro experimentation, animals were transferred 3 to 4 weeks later to a housing room with identical conditions, 3–5 days prior to sacrifice. For in vivo experimentation, animals were brought to the recording room at least one week prior to experimentation. All experimental procedures complied with the Swiss National Institutional Guidelines on Animal Experimentation and were approved by the Swiss Cantonal Veterinary Office Committee for Animal Experimentation.
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