HEBf E2Af 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.
Yuan Zhuang, Duke University Medical Center
Genetic Background | Generation |
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|
Allele Type | Gene Symbol | Gene Name |
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Targeted (Conditional ready (e.g. floxed), No functional change) | Tcf3 | transcription factor 3 |
Allele Type | Gene Symbol | Gene Name |
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Targeted (Conditional ready (e.g. floxed), No functional change) | Tcf12 | transcription factor 12 |
These HEBf E2Af mice harbor two mutations, the HEB floxed allele (Tcf12tm3Zhu) and E2A floxed allele (Tcf3tm4Zhu); 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 E2Af allele also expresses β-galactosidase (lacZ) following exposure to Cre recombinase. Specific examples are described below.
HEBf E2Af 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.
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).
Allele Name | targeted mutation 4, Yuan Zhuang |
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Allele Type | Targeted (Conditional ready (e.g. floxed), No functional change) |
Allele Synonym(s) | E2Aloxp |
Gene Symbol and Name | Tcf3, transcription factor 3 |
Gene Synonym(s) | |
Strain of Origin | 129S4/SvJaeSor |
Chromosome | 10 |
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. |
Mutations Made By | Yuan Zhuang, Duke University Medical Center |
Allele Name | targeted mutation 3, Yuan Zhuang |
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Allele Type | Targeted (Conditional ready (e.g. floxed), No functional change) |
Allele Synonym(s) | HEBflox; Tcf12f |
Gene Symbol and Name | Tcf12, transcription factor 12 |
Gene Synonym(s) | |
Strain of Origin | Not Specified |
Chromosome | 9 |
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. |
These HEBf E2Af mice harbor two mutations that segregate independently; the HEB floxed allele (Tcf12tm3Zhu) and E2A floxed allele (Tcf3tm4Zhu). When maintaining a live colony, mice homozygous for both floxed mutations may be bred together.
When using the HEBflox E2Aflox mouse strain in a publication, please cite the originating article(s) and include JAX stock #024511 in your Materials and Methods section.
Facility Barrier Level Descriptions
Service/Product | Description | Price |
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Heterozygous for Tcf12<tm3Zhu>, Heterozygous for Tcf3<tm4Zhu> |
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