Skip to main content
. 2020 Mar 25;204(9):2600–2611. doi: 10.4049/jimmunol.2000025

FIGURE 1.

FIGURE 1.

Generation of Gata3 knock-in reporter (GATIR) mice that can be maintained as a homozygous line. (A) Targeting strategy. TK indicates the gene encoding herpes simplex thymidine kinase; NEO indicates the neomycin resistance-encoding gene. 3′-UT indicates the 3′-untranslated region of the Gata3 gene; IRES (stippled box) indicates the IRES; vYFP (green box) indicates the vYFP-encoding cDNA. (B) Multiplex RT-PCR reveals robust expression of the targeted allele and confirms insertion of the reporter cassette at the predicted site. Left panel, Exon assignment of the Gata3 cDNA template (partial), including the position of the three primers (P1, P2, and P3) used for multiplex RT-PCR analysis. P1 anneals in exon 4–encoded sequences, P2 anneals within a short artificial linker region at the 5′-end of the IRES cassette, and P3 anneals in the 3′-untranslated region downstream of the IRESvYFP insertion site. The dotted lines below indicate the predicted fragment size for transcripts from wild-type (WT) and knock-in (KI) alleles, respectively; right panel: result of a representative RT-PCR analysis with total RNA isolated from unfractionated thymocytes of two different WT (WT/WT), heterozygous (KI/WT), and homozygous (KI/KI) GATIR mice, respectively. Please note that primer combination P1/P3 does not give rise to an amplified PCR product with cDNA derived from the KI allele under our RT-PCR conditions, which were optimized for amplification products <1 kb. The theoretical P1/P3–primed amplification product from message of the targeted allele would be 2.24 kb. M indicates the DNA size marker. (C) Genotype distribution of KI and WT alleles among offspring of heterozygous GATIR breeders. Data were obtained with a total of 49 litters from 16 independent breeding cages over a period of 2 y (July 17, 2017–July 2, 2019). Genotyping was performed at the time of weaning (i.e., 4–5 wk after birth). (D) There was no difference in average litter size among offspring of homozygous GATIR versus WT C57BL/6 breeders. Breeding performance was monitored over a period of ∼15 mo (January 1, 2017–April 10, 2018) and determined at the time of weaning. Homozygous GATIR breeders were on 20th generation C57BL/6 backcross. Breeders of both genotypes (GATIR and WT C57BL/6) were housed in the same room of our animal facility.