These HEBf E2Af mice harbor two mutations; the HEB floxed allele (Tcf12tm3Zhu) and E2A floxed allele (Tcf3tm4Zhu).
The HEBflox (or HEBf) targeting construct was designed by Dr. Yuan Zhuang (Duke University Medical Center) to insert a loxP site upstream of the basic helix-loop-helix encoding sequence (exon 18), as well as a loxP-flanked PGK-neo cassette downstream of exon 18 of the transcription factor 12 gene (Tcf12) on chromosome 9. The construct was electroporated into undisclosed embryonic stem (ES) cells. Correctly targeted ES cells were transiently transfected with a Cre-recombinase expression vector. ES cells retaining the floxed exon 18 and with the PGK-neo removed were identified and injected into recipient blastocysts. The chimeric animals were bred to undisclosed mice to establish the HEBf colony. The donating investigator reports that the HEBf colony was backcrossed with C57BL/6J more than ten generations (see SNP note below). The resulting C57BL/6J-congenic HEBf mice were then used as described below.
The E2Aloxp (or E2Af) targeting construct was designed by Dr. Yuan Zhuang (Duke University Medical Center) to insert a loxP site into an intron upstream of the basic helix-loop-helix encoding sequence (common to both E12 and E47 isoforms), as well as a PGK-neo, loxP site, splice acceptor site, IRES-lacZ and transcription stop signal downstream of the coding region of the transcription factor 3 gene (Tcf3) on chromosome 10. The construct was electroporated into 129S4/SvJaeSor-derived AK7 embryonic stem (ES) cells. Correctly targeted ES cells were injected into recipient blastocysts and chimeric animals were bred to undisclosed mice to establish the E2Af colony. The donating investigator reports that the E2Af colony was backcrossed with C57BL/6J more than ten generations (see SNP note below). The resulting C57BL/6J-congenic E2Af mice were then used as described below.
To generate the double floxed line, Dr. Zhuang bred C57BL/6J-congenic HEBf mice with C57BL/6J-congenic E2Af mice (see SNP note below). The HEBf E2Af mice were sent to The Jackson Laboratory Repository in 2014. Upon arrival, males were used to cryopreserve sperm. To establish our living mouse colony, an aliquot of the frozen sperm was used to fertilize oocytes from C57BL/6J inbred females (Stock No. 000664).
In 2015, a 32 SNP (single nucleotide polymorphism) panel analysis, with 27 markers covering all 19 chromosomes and the X chromosome, as well as 5 markers that distinguish between the C57BL/6J and C57BL/6N substrains, was performed on the first generation rederived living colony at The Jackson Laboratory Repository. This revealed 4 of 27 markers were not fixed for C57BL/6 allele-type (i.e., still segregating for 129S1 allele-type markers on chromosomes 3 [~37.3 Mbp], 8 [~14.8 Mbp], 11 [~20.9 Mbp] and 12 [~30.7 Mbp]).
In addition, 4 of the 5 markers that determine C57BL/6J from C57BL/6N revealed some mice were segregating for 129S1 allele-type markers (chromosomes 8 [~15.2 Mbp], 11 [~4.4 Mbp], 15 [~57 Mbp] and 19 [~50 Mbp]), and the 5th marker was C57BL/6J.
Collectively, these data suggest the mice sent to The Jackson Laboratory were on a mixed genetic background (~77% C57BL/6).
