STOCK Tcf12^tm3Zhu Tcf3^tm4Zhu/J

Stock number: 024511
Product name: HEB^flox E2A^flox
Research areas: Cancer Research, Developmental Biology Research, Hematological Research, Immunology, Inflammation and Autoimmunity Research, Internal/Organ Research, Neurobiology Research, Research Tools, Virology Research

Description

HEB^f E2A^f mice allow Cre-recombinase inducible deletion of the two basic helix-loop-helix (bHLH) E-protein family genes HEB and E2A. These mice may be useful for studying B and T lymphocyte development and lymphoid malignancies.

Detailed description

These HEB^f E2A^f mice harbor two mutations, the HEB floxed allele (Tcf12^tm3Zhu) and E2A floxed allele (Tcf3^tm4Zhu); each with its basic helix-loop-helix domain flanked by loxP sites.

The transcription factors E2A and HEB (members of the E protein family) have been shown to play essential roles in lymphocyte development, and mutations/deficiencies are associated with lymphoid malignancies. Mice homozygous for both floxed alleles are viable and fertile with no reported abnormalities. When bred to mice that express Cre recombinase, the resulting offspring may be useful in generating tissue-specific alterations in B cell and T cell development. The E2A^f allele also expresses β-galactosidase (lacZ) following exposure to Cre recombinase. Specific examples are described below.

HEB^f E2A^f mice may be bred to animals expressing Cre recombinase in different stages of T cell development. When bred to mice expressing Cre recombinase in the very late double-negative stage of T cell development (CD4-Cre transgenic mice; see Stock No. 017336), the resulting mice allow knockout of these two genes before the double-positive stage of T-cell development. When bred to mice expressing Cre recombinase later in T cell development (such as Lck-Cre transgenic mice; Stock No. 012837), the resulting mice allow knockout of these two genes after positive selection in the thymus.

Development

These HEB^f E2A^f mice harbor two mutations; the HEB floxed allele (Tcf12^tm3Zhu) and E2A floxed allele (Tcf3^tm4Zhu).

The HEB^flox (or HEB^f) 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 HEB^f colony. The donating investigator reports that the HEB^f colony was backcrossed with C57BL/6J more than ten generations (see SNP note below). The resulting C57BL/6J-congenic HEB^f mice were then used as described below.

The E2A^loxp (or E2A^f) 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 E2A^f colony. The donating investigator reports that the E2A^f colony was backcrossed with C57BL/6J more than ten generations (see SNP note below). The resulting C57BL/6J-congenic E2A^f mice were then used as described below.

To generate the double floxed line, Dr. Zhuang bred C57BL/6J-congenic HEB^f mice with C57BL/6J-congenic E2A^f mice (see SNP note below). The HEB^f E2A^f 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).

Allele

Symbol: Tcf12^tm3Zhu
Name: targeted mutation 3, Yuan Zhuang
MGI accession ID: MGI:3769702
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed); No functional change
Marker symbol: Tcf12
Marker name: transcription factor 12
Chromosome: 9
Strain of origin: Not Specified
Molecular note: A floxed neo cassette was inserted into intron 18 and an additional loxP site was inserted into intron 17. The neo cassette was removed by cre-mediated recombination using a transiently expressing cre vector leaving a floxed exon 18.

Allele

Symbol: Tcf3^tm4Zhu
Name: targeted mutation 4, Yuan Zhuang
MGI accession ID: MGI:2387315
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed); No functional change
Marker symbol: Tcf3
Marker name: transcription factor 3
Chromosome: 10
Strain of origin: 129S4/SvJaeSor
Molecular note: A targeting construct was engineered to contain a 5' loxP site in an intron upstream of the bHLH sequence, common to both E12 and E47 isoforms, and a 3' loxP site downstream of the endogenous locus. Also included in the construct were neo and lacZ genes, respectively positioned upstream and downstream of the 3' loxP site. The construct was designed such that the lacZ gene will be transcribed only upon recombination of the loxP sites.