BKa.Cg-Ptprc^b Bmi1^tm1Ilw Thy1^a/J

Stock number: 017351
Product name: Bmi-1GFP
Research areas: Cell Biology Research, Developmental Biology Research, Hematological Research, Immunology, Inflammation and Autoimmunity Research, Research Tools

Description

The Bmi-1^GFP 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 (Ptprc^b) and Thy1.1 leukocyte alloantigen (Thy1^a) alleles to allow tracking of hematopoietic donor derived or transferred cells.

Detailed description

The Bmi-1^GFP 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-1^GFP 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 GFP^high and GFP^lo show that the Bmi-1 expression level in the GFP^high population is approximately 10-fold higher than that of the GFP^lo population; these Bmi-1^high cell populations are functional HSCs.

The donating investigator reports that mice homozygous for the Bmi-1^GFP 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 Ptprc^b 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, Thy1^a, 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 Thy1^a serves as a marker for tracking donor cells in vitro.

Development

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-1^GFP 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-Ptprc^b Thy1^a) to generate the colony. The Bmi-1^GFP 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-1^GFP::CD45.2::Thy1.1 males were sent to The Jackson Laboratory Repository. Upon arrival, these mice were bred together to establish the colony.

Allele

Symbol: Bmi1^tm1Ilw
Name: targeted mutation 1, Irving L Weissman
MGI accession ID: MGI:3806975
Generation method: Targeted
Attributes: Reporter; Null/Knockout
Marker symbol: Bmi1
Marker name: Bmi1 polycomb ring finger oncogene
Chromosome: 2
Strain of origin: (129X1/SvJ x 129S1/Sv)F1-Kitl⁺
Expressed genes: GFP,Green Fluorescent Protein,
Site of expression: EGFP expression (direct fluorescence) is highest in hematopoietic stem cell (HSC) populations and downregulated along with differentiation.
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.

Allele

Symbol: Ptprc^b
Name: b variant
MGI accession ID: MGI:4819850
Generation method: Not Applicable
Marker symbol: Ptprc
Marker name: protein tyrosine phosphatase, receptor type, C
Chromosome: 1
Strain of origin: Not Applicable
Site of expression: Widely expressed on all adaptive and innate immune cells.
Molecular note: Ptprc^b 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).

Allele

Symbol: Thy1^a
Name: a variant
MGI accession ID: MGI:3579311
Generation method: Not Applicable
Marker symbol: Thy1
Marker name: thymus cell antigen 1, theta
Chromosome: 9
Strain of origin: Not Applicable
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.
Molecular note: The allele Thy1^a determines an antigenic specificity, Thy-1.1, found in the AKR and RF strains.
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 Thy1^a determines an antigenic specificity, Thy-1.1, found in the AKR and RF strains; the allele Thy1^b 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).