Lrig1T2A-iCreERT2 mouse line 2f1

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Lrig1T2A-iCreERT2 mouse line 2f1
Autor: Nam, Hyung-song ; Capecchi, Mario
Assuntos: Neurogenetics
Notas: https://link.springer.com/article/10.1186/s13064-020-00139-5
https://link.springer.com/article/10.1186/s13064-022-00169-1
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7077007/
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9832784/
Descrição: Two Lrig1T2A-iCreERT2 lines were generated from two ESC clones. Line 1c2 had a postnatal lethality problem (see here). Thus, line 2f1 was also characterized.The Lrig1T2A-iCreERT2 allele design. The Lrig1T2A-iCreERT2 allele targeting vector map. The vector was recombineered from a C57BL/6J BAC library clone (NGS sequence of the vector here).Characterization of the sfGFP-iCre-ERT2. (1) A test of the mutated P2A between sfGFP and iCre-ERT2 in Lrig1T2A-iCreERT2 allele for ribosome skip activity. See here for the rationale behind using fusions to histone H2B-sfGFP. Basically, without ribosome skip, the tdTomato fluoresces in the nucleus, which is what one sees in these images. (2) Activity and dim fluorescence of the sfGFP-iCre-ERT2 fusion protein. For additional data on the activity of the sfGFP-iCre-ERT2 fusion protein, see here. For additional data on the in vivo fluorescence of the sfGFP-iCre-ERT2 fusion protein, see here and here.To generate the mice, a stem cell line G4 from a C57BL/6Ncr x 129S6/SvEvTac F1 mixed genetic background mouse embryo was targeted with the vector above. Mice were generated from two stem cell clones, 1c2 and 2f1. Germline 1c2 mice were crossed to mixed genetic background Tg(ACTB-FLPe) mice to loop out the Neo then repeatedly backcrossed to C57BL/6J background while breeding out the FLPe transgene and fixing the X and Y chromosomes to the C57BL/6J background. Germline 2f1 mice were crossed to C57BL/6J congenic Tg(Pgk1-FLPo)/+ mice (Simon Titen, Capecchi laboratory) then also repeatedly backcrossed to the C57BL/6J background as for the 1c2 mice (also see here for more information).Notes on PCR genotyping of mouse lines. Works as is (i.e., the total reaction volume is off by a microliter because the amount of the DNA was doubled to prevent pipetting errors).2308 is a routine PCR genotyping gel of Lrig1T2A-iCreERT2 allele and Lrig1T2A-tdTomato allele mice from backcrosses to the C57BL/6J mice. The two alleles are genotyped with the same primers, and show the same size products.A subline of the 2f1 Lrig1T2A-iCreERT2 homozygotes was generated with intercross of het mice from backcross generation N6. 1941 shows in red box genotyping PCR of weanlings from the 2f1 line N6 het intercross. Bottom band is the knock-in band. Top band is the wildtype band. This lineage of mice was used exclusively for Nam and Capecchi, 2023. They continued to breed without any problems and were discontinued at generation F8 around June 2022.Separate from the homozygous subline generated from the N6 het mice intercross, the backcross of the line 2f1 mice to the C57BL/6J background was continued until generation N12 then the mice intercrossed. 2367 shows in red box genotyping PCR of weanlings from the N12 het intercross. The N12 het intercross also generated homozygotes at Mendelian ratio. The resultant homozygous mice continued to breed without any problems and were discontinued at generation F4 around May 2023. Line 2f1 heterozygous mice of the backcross generation N12 were shipped to the MMRRC on April 18, 2022.Line 2f1 breeding history.Summary in high resolution pdf file.Videos of some C57BL/6J-backcrossed wildtype mice from a different colony and C57BL/6J-backcrossed 2f1 Lrig1T2A-iCreERT2 homozygous mice (generation F3 of homozygotes from the N12 het intercross). All were 7 week-old males.Whole mount immunofluorescence for GFAP, DCX, KI-67, and ASCL1 on lateral ventricular walls from the 7 week-old wildtype and Lrig1T2A-iCreERT2 homozygous mice shown above (both backcrossed to the C57BL/6J background). Top row shows 3 wildtype samples. Bottom row shows 3 homozygote samples. I don't see any obvious differences in the proliferation markers between the wildtype and homozygous knock-in mice samples.Dosage analysis using Laboras. The Lrig1T2A-iCreERT2 homozygous mice were older littermates (12 weeks of age) of the F3 2f1 mice shown above. The Lrig1T2A-iCreERT2 heterozygous and trans heterozygous mice were generated with the line 1c2 mice. As noted in Nam and Capecchi, 2020, differences in rearing and distance traveled were statistically signficant between Lrig1 wildtype and littermate knock-out mice. Importantly, there were no differences in these values between C57BL/6J mice and the C57BL/6J-backcrossed Lrig1T2A-iCreERT2 homozygous mice (line 2f1) as well as the trans heterozygous mice (line 1c2, also see here). Taken together with the proliferation marker analysis above, it can be concluded that the Lrig1 knock-in did not alter the locus function.Some cage cards of F4 generation mice from breeding F3 males and F3 females. Normal size litters were weaned from the homozygous males and females. Acknowledgment to Dr. Anne Boulet for taking the photographs in my absence (because I left Salt Lake City on 4/22/2023).✴︎Videos of 2f1 Lrig1T2A-iCreERT2/+; Rosa26Ai14/+ mice at different ages.To generate the mice below, homozygous Lrig1T2A-iCreERT2/T2A-iCreERT2 males from the N6 het intercross and Rosa26Ai14/Ai14 females were bred. The Rosa26Ai14/+ mice had been backcrossed seven times to the C57BL/6J background and homozygosed (Simon Titen, Capecchi laboratory)."12 week old males, TMX at 3 weeks *.mov" are 12 week-old 3 males. Injected 1x 120 mg/kg tamoxifen at 3 weeks."14 week old females, TMX at 13 weeks *.mov" are 14 week-old 4, 2, and 3 females. Injected 1x 214 mg/kg tamoxifen at 13 weeks. The Lrig1T2A-iCreERT2 activity in the line 2f1 mice (see here for more)."7 month old *.mov" are ~7 month-old 3 males and 3 females. Not injected tamoxifen."10 month old *, TMX at 12 weeks.mov" are ~10 month-old 2 males and 2 females. Injected 1x 214 mg/kg tamoxifen at 12 weeks. If you think the tails and ears show a tint of red color, that's probably because of the RFP in the skin."1 year 3 month old *.mov" are ~1 year and 3 month old 3 males and 4 females. Not injected tamoxifen."2 year old *.mov" are ~2 year old 2 males, 4 males, 3 females, and 1 female (a cage mate had died). Not injected tamoxifen. At this age, some are obese due to the C57BL/6J genetic background and the diet.✼The 2f1 Lrig1T2A-iCreERT2 mice will be available from MMRRC (069516-UCD).The Lrig1T2A-tdTomato mice will be available from Jax (# 036457).We refer interested investigators to MMRRC and Jax to obtain these mouse lines.
Editor: figshare
Data de criação/publicação: 2024
Idioma: Inglês

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Lrig1T2A-iCreERT2 mouse line 2f1

Nam, Hyung-song ; Capecchi, Mario

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