B6;129-Pou2f2^{tm1c(EUCOMM)Hmgu}/SdtJ

Stock number: 029132
Product name: Pou2f2^fl (OCT2^fl)
Research areas: Cancer Research, Immunology, Inflammation and Autoimmunity Research, Research Tools

Description

The "Cre-conditional ready (post-Flp)" Pou2f2^{tm1c(EUCOMM)Hmgu} allele (Pou2f2^fl or OCT2^fl) has loxP sites flanking exons 8-11 (encoding the entire POU domain). Exposure to Cre recombinase will delete exons 8-11 and result in the knockout allele. These mice may be useful in studying B cell function, humoral immunity and cancers such as human diffuse large B-cell lymphoma.

Detailed description

Expression of the transcription factor octamer-binding protein 2 (OCT2; encoded by Pou2f2) is largely restricted to the B-cell lineage; where it functions in the later stages of B-cell differentiation and promotes the proliferation and plasmacytic differentiation of germinal center B cells. In addition, OCT2 is highly expressed in normal germinal center B cells and in almost all cases of human diffuse large B-cell lymphoma (DLBCL).

The "Cre-conditional ready (post-Flp)" Pou2f2^{tm1c(EUCOMM)Hmgu} allele (Pou2f2^fl or OCT2^fl) has loxP sites flanking exons 8-11 (encoding the entire POU domain). Before exposure to Cre recombinase, the Pou2f2^fl allele expresses a normal Pou2f2 transcript encoding the functional Pou2f2 protein. Mice heterozygous (Pou2f2^fl/wt) or homozygous (Pou2f2^fl/fl) for this floxed allele are viable and fertile, with no reported gross phenotypic abnormalities or breeding problems.

Because Pou2f2^fl is a "Cre-conditional ready (post-Flp)" allele, exposure to Cre recombinase will delete exons 8-11 and result in the knockout allele.
Specifically, breeding Pou2f2^fl to have to ROSA26-CreERT2 (see Stock No. 008463) allows tamoxifen-inducible OCT2-deficiency in germinal center B cells and in splenic B cells. The phenotype of such tamoxifen-induced OCT2-deficient (homozygous) adult mice is described below.
The resulting knockout allele produces a Pou2f2 transcript that is translated into an unstable protein lacking exons 8-11. While other mutant mice with germline Pou2f2-deficiency die shortly after birth, the OCT2-knockout induced by tamoxifen injection in these adult mice does not lead to health defects or altered behavior (out to 2 months after deletion). These OCT2-deficient mice exhibit an abnormal response to antigen immunization: although able to initiate normal germinal center B cell formation and produce normal numbers of memory B cells, they have reduced proliferation of B cells within the germinal center and no ability to differentiate into produce antibody-secreting plasma cells.

Development

The "Cre-conditional ready (post-Flp)" Pou2f2^{tm1c(EUCOMM)Hmgu} allele (Pou2f2^fl or OCT2^fl) has loxP sites flanking exons 8-11 (encoding the entire POU domain).

The details for the tm1a allele (prior to germline FLP expression) are shown on International Knockout Mouse Consortium/International Mouse Phenotyping Consortium (IMPC) website: IKMC Project 82506 Pou2f2^{tm1a(EUCOMM)Hmgu} "knockout first (reporter-tagged insertion with conditional potential)" allele.

Specifically, the L1L2_Bact_P cassette was placed upstream of exons 8 of the POU domain, class 2, transcription factor 2 locus (Pou2f2); insertion at bp position Chr7:25,098,260 [Build GRCm38]. The L1L2_Bact_P cassette is composed of an frt site, a mouse En2 intron/splice-acceptor/exon (En2 SA), an IRES sequence, the β-galactosidase gene (lacZ) with SV40 polyadenylation signal (SV40pA), a loxP site, the human beta actin promoter-driven neomycin-resistance gene with SV40 polyadenylation signal (hbactP-neo-pA), a second frt site and a second loxP site. A third loxP site was inserted downstream of exon 11 at bp position Chr7:25,096,600 [Build GRCm38]; therefore flanking the critical exons (exons 8-11) with loxP sites.
The targeting vector PG00212_Y_7_D08 was introduced into C57BL/6N-A^tm1Brd-derived JM8A3.N1 embryonic stem (ES) cells. Dr. Louis M. Staudt (NIH/NCI) obtained the correctly targeted ES cell clone HEPD0690_4_H11 from the International Knockout Mouse Consortium/International Mouse Phenotyping Consortium (IMPC), and injected them into recipient blastocysts. Chimeric males were bred with albino C57BL/6 females (C57BL/6-cBrd/cBrd/Cr) for germline production. Next, mice were bred with B6.Cg-Tg(ACTFLPe)9205Dym/J (Stock No. 005703) for germline removal of the frt-flanked splice-acceptor::IRES::lacZ-loxP-neo cassette. The donating investigator reports that neither the albino allele nor the FLP transgene was retained. The Pou2f2^fl colony was also bred to ROSA26-CreER mice on a 129 or C57BL/6;129 genetic background (Gt(ROSA)26Sor^{tm1(cre/Esr1)Tyj}). Thereafter, the Pou2f2^fl colony was maintained by breeding homozygotes with heterozygotes for several generations. A single backcross to C57BL/6J mice was also used to remove the ROSA26-CreER allele. Heterozygous males with black coat color will be sent to The Jackson Laboratory Repository. Upon arrival, sperm was cryopreserved. To establish our live colony, an aliquot of frozen sperm was used to fertilize C57BL/6J oocytes (Stock No. 000664).

For additional information on all KOMP targeting strategies, see KOMP International Mouse Phenotyping Consortium website about KOMP targeting strategies.

In 2017, a 48 SNP (single nucleotide polymorphism) panel analysis, with 43 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 males sent to The Jackson Laboratory Repository. While the 43 markers throughout the genome suggested a C57BL/6 genetic background, all 5 markers that determine C57BL/6J from C57BL/6N were found to be segregating. These data show the mice sent to The Jackson Laboratory Repository were on a mixed C57BL/6N;C57BL/6J genetic background.

Allele

Symbol: Pou2f2^{tm1c(EUCOMM)Hmgu}
Name: targeted mutation 1c, Helmholtz Zentrum Muenchen GmbH
MGI accession ID: MGI:5767180
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed); No functional change
Marker symbol: Pou2f2
Marker name: POU domain, class 2, transcription factor 2
Chromosome: 7
Strain of origin: C57BL/6N-A^tm1Brd
Molecular note: The L1L2_Bact_P cassette was inserted at position 25098260 of Chromosome 7 upstream of the critical exons 8 through 11 (Build GRCm38). The cassette is composed of an FRT site followed by lacZ sequence and a loxP site. This first loxP site is followed by neomycin resistance gene under the control of the human beta-actin promoter, SV40 polyA, a second FRT site and a second loxP site. A third loxP site is inserted downstream of the targeted exons 8 through 11 at position 25096600. The critical exons 8 through 11 are thus flanked by loxP sites. A "conditional ready" (floxed) allele was created by flp recombinase expression in mice carrying this allele. Subsequent cre expression results in a knockout mouse. If cre expression occurs without flp expression, a reporter knockout mouse will be created. Further information on targeting strategies used for this and other IKMC alleles can be found at http://www.informatics.jax.org/mgihome/nomen/IKMC_schematics.shtml.