The Bmi-1GFP knock-in/knock-out allele was designed to both abolish Bmi1 gene function and express EGFP from the Bmi1 promoter/enhancer elements. Of note, these mice also harbor the CD45.2 (Ptprcb) and Thy1.1 leukocyte alloantigen (Thy1a) alleles to allow tracking of hematopoietic donor derived or transferred cells.
Irving L Weissman, Stanford University
Genetic Background | Generation |
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|
Allele Type | Gene Symbol | Gene Name |
---|---|---|
Not Applicable | Thy1 | thymus cell antigen 1, theta |
Allele Type | Gene Symbol | Gene Name |
---|---|---|
Targeted (Reporter, Null/Knockout) | Bmi1 | Bmi1 polycomb ring finger oncogene |
Allele Type | Gene Symbol | Gene Name |
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Not Applicable | Ptprc | protein tyrosine phosphatase, receptor type, C |
The Bmi-1GFP knock-in/knockout allele has EGFP fused in-frame with the endogenous transcriptional start site of the Bmi-1 polycomb ring finger oncogene (Bmi1) locus; both abolishing endogenous gene function and placing EGFP expression under direction of the Bmi1 promoter/enhancer regions. EGFP expression (direct fluorescence) is highest in hematopoietic stem cell (HSC) populations and downregulated along with differentiation; recapitulating the expected expression pattern of endogenous Bmi-1. Specifically, EGFP fluorescence is significantly higher in the HSC-enriched KLS population (c-kit+lin-Sca-1+) compared with other populations.
Mice heterozygous for the Bmi-1GFP knock-in/knockout allele are viable, fertile, and phenotypically indistinguishable from wildtype littermate controls with respect to survival, hematopoietic cellularity, and lineage composition. Heterozygous bone marrow cells sorted via FACS into GFPhigh and GFPlo show that the Bmi-1 expression level in the GFPhigh population is approximately 10-fold higher than that of the GFPlo population; these Bmi-1high cell populations are functional HSCs.
The donating investigator reports that mice homozygous for the Bmi-1GFP knock-in/knockout allele die before birth or soon after they are born. In addition, homozygotes may be expected to exhibit the same phenotype as mice homozygous for other null mutations of this gene (decreased hematopoietic cell number, immune deficiency, neurological abnormalities, and posterior transformation).
These mice also harbor the Ptprcb allele, CD45.2, of C57BL/6J origin rather than the alternate allele normally present in C57BL/Ka inbred mice. As CD45 is expressed on all adaptive and innate immune cells, this strain provides a unique cell surface marker that can be used to track lineage and phenotype of all hematopoietic donor derived or transferred cells.
In addition, these mice also harbor the Thy1.1 leukocyte alloantigen allele, Thy1a, rather than the alternate allele normally present in C57BL/Ka inbred mice (as well as C57BL/6, C57BL/10, DBA/2, NOD, and BALB/c inbred mice). Thus, cell populations derived from these mutant mice can be distinguished from syngeneic host and other mice with the alternate allele via flow cytometry. The presence of Thy1a serves as a marker for tracking donor cells in vitro.
A targeting vector was designed to replace exon 2 of the Bmi1 polycomb ring finger oncogene (Bmi1) locus with an enhanced green fluorescent protein (EGFP) and loxP-flanked neomycin resistance cassette. The EGFP is inserted in-frame to the Bmi1 ATG translation start site. The construct was electroporated into (129X1/SvJ x 129S1/Sv)F1-Kitl+)-derived R1 embryonic stem (ES) cells. Correctly targeted ES cells were then transiently transfected with a Cre recombinase-expressing plasmid. The resulting ES cells with the Bmi-1GFP genotype (neo selection cassette removed; leaving EGFP and a single loxP site replacing exon 2) were injected into recipient blastocysts. Chimeric animals were bred to C57BL/Ka mice with the CD45.2 and Thy1.1 alleles (C57BL/Ka-Ptprcb Thy1a) to generate the colony. The Bmi-1GFP mutant mice were backcrossed to the C57BL/Ka-CD45.2::Thy1.1 line for more than twenty generations. C57BL/Ka-CD45.2::Thy1.1 females and C57BL/Ka-Bmi-1GFP::CD45.2::Thy1.1 males were sent to The Jackson Laboratory Repository. Upon arrival, these mice were bred together to establish the colony.
Expressed Gene | GFP, Green Fluorescent Protein, |
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Site of Expression | EGFP expression (direct fluorescence) is highest in hematopoietic stem cell (HSC) populations and downregulated along with differentiation. |
Site of Expression | Widely expressed on all adaptive and innate immune cells. |
Allele Name | a variant |
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Allele Type | Not Applicable |
Allele Synonym(s) | thetaAKR; theta-AKR; Thy1.1; Thy-1.1; Thy1a |
Gene Symbol and Name | Thy1, thymus cell antigen 1, theta |
Gene Synonym(s) | |
Site of Expression | The Thy1 locus determines a surface antigen present on cells of the thymus, a number of mouse leukemias, brain, and in lesser amounts on lymph node and spleen cells. |
Strain of Origin | Not Applicable |
Chromosome | 9 |
General Note | The Thy1 locus determines a surface antigen present on cells of the thymus, a number of mouse leukemias, brain, and in lesser amounts on lymph node and spleen cells. The allele Thy1a determines an antigenic specificity, Thy-1.1, found in the AKR and RF strains; the allele Thy1b determines an antigenic specificity, Thy-1.2, found in the C3HeB/Fe and many other strains (J:5243, J:5012, J:4469). The Thy1 antigen is probably present on all T lymphocytes and absent from all B lymphocytes, and it thus serves as a valuable T-cell marker (J:5243). It is very widely used in experiments designed to determine the distribution and function of T-cells. Thy1 specifies a T-cell surface glycoprotein, T25, with a molecular weight of 25 kDa (J:5707). The protein appears to be anchored in the cell membrane by a lipid that is either phosphotidylinositol or closely related to it (J:12016). Thy1 may function in the cell membrane as a signal transduction molecule (J:8333). The Thy1 locus, or possibly a gene closely linked to it, controls quantitative expression of a protein that is the same size as Thy1 and is expressed on thymus and brain but not on lymph node and spleen cells (J:7900). |
Molecular Note | The allele Thy1a determines an antigenic specificity, Thy-1.1, found in the AKR and RF strains. |
Allele Name | targeted mutation 1, Irving L Weissman |
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Allele Type | Targeted (Reporter, Null/Knockout) |
Allele Synonym(s) | Bmi-1GFP; Bmi1tm1Nhsn |
Gene Symbol and Name | Bmi1, Bmi1 polycomb ring finger oncogene |
Gene Synonym(s) | |
Expressed Gene | GFP, Green Fluorescent Protein, |
Site of Expression | EGFP expression (direct fluorescence) is highest in hematopoietic stem cell (HSC) populations and downregulated along with differentiation. |
Strain of Origin | (129X1/SvJ x 129S1/Sv)F1-Kitl+ |
Chromosome | 2 |
Molecular Note | Exon 2 was replaced with an in frame EGFP cassette, exon 2 and floxed neo cassette. Transient cre expression in ES cells was used to remove the neo cassette. Expression of the EGFP precludes expression of the endogenous gene. |
Mutations Made By | Irving Weissman, Stanford University |
Allele Name | b variant |
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Allele Type | Not Applicable |
Allele Synonym(s) | CD45.2; Ly5b |
Gene Symbol and Name | Ptprc, protein tyrosine phosphatase, receptor type, C |
Gene Synonym(s) | |
Site of Expression | Widely expressed on all adaptive and innate immune cells. |
Strain of Origin | Not Applicable |
Chromosome | 1 |
Molecular Note | Ptprcb is found in strains C57BL/6, C3H/An, DBA/2, AKR, and many others (J:13367, J:12054, J:12077, J:8603). Twelve nucleotide differences between the a and b alleles have been identified. These base substitutions correspond to five amino-acid changes within the extracellular domain of the encoded protein. These amino-acid differences are clustered in a region that also contains the greatest divergence between mouse and rat sequences (J:22485). Note that the allele designations originally described were reversed in 1987 (J:8603); all publications prior to 1987 show SJL/J, STS/A, and DA as having the b allele and the C57BL/6J group as having the a allele (J:22341). |
When maintaining a live colony, mice heterozygotes for the Bmi1GFP mutation and homozygous for both the Ptprcb and Thy1a alleles are bred with mice from the colony that are wildtype at the Bmi1 locus and homozygous for both the Ptprcb and Thy1a alleles. Mice homozygous for the Bmi1GFP mutation die before birth or soon after they are born.
When using the Bmi-1GFP mouse strain in a publication, please cite the originating article(s) and include JAX stock #017351 in your Materials and Methods section.
Facility Barrier Level Descriptions
Service/Product | Description | Price |
---|---|---|
Homozyogus for Ptprc<b>,Heterozygous or wildtype for Bmi1<tm1Ilw>, Homozygous for Thy1<a> |
Frozen Mouse Embryo | BKa.Cg-Ptprc<b> Bmi1<tm1Ilw> Thy1<a>/J Frozen Embryos | $2595.00 |
Frozen Mouse Embryo | BKa.Cg-Ptprc<b> Bmi1<tm1Ilw> Thy1<a>/J Frozen Embryos | $2595.00 |
Frozen Mouse Embryo | BKa.Cg-Ptprc<b> Bmi1<tm1Ilw> Thy1<a>/J Frozen Embryos | $3373.50 |
Frozen Mouse Embryo | BKa.Cg-Ptprc<b> Bmi1<tm1Ilw> Thy1<a>/J Frozen Embryos | $3373.50 |
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The Jackson Laboratory has rigorous genetic quality control and mutant gene genotyping programs to ensure the genetic background of JAX® Mice strains as well as the genotypes of strains with identified molecular mutations. JAX® Mice strains are only made available to researchers after meeting our standards. However, the phenotype of each strain may not be fully characterized and/or captured in the strain data sheets. Therefore, we cannot guarantee a strain's phenotype will meet all expectations. To ensure that JAX® Mice will meet the needs of individual research projects or when requesting a strain that is new to your research, we suggest ordering and performing tests on a small number of mice to determine suitability for your particular project. We do not guarantee breeding performance and therefore suggest that investigators order more than one breeding pair to avoid delays in their research.
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