B6;129-Prg4^tm2Mawa/J

Stock number: 025740
Research areas: Immunology, Inflammation and Autoimmunity Research, Research Tools

Description

These mice carry a floxed/reversible gene trap allele of the mouse Prg4 gene designed to produce membrane-anchored β-galactosidase instead of lubricin in cells where Prg4 is normally expressed. Cre-mediated excision of the genetrap restores lubricin expression.

Detailed description

Congenital deficiency of the principal boundary lubricant in cartilage (lubricin, encoded by the gene Prg4, proteoglycan 4 (megakaryocyte stimulating factor, articular superficial zone protein)) increases joint friction and causes progressive joint failure.

These mice carry a floxed gene trap allele of the mouse Prg4 gene designed to produce membrane-anchored β-galactosidase instead of lubricin in cells where Prg4 is normally expressed. The genetrap allele does not express wildtype Prg4 mRNA, as determined by RT-PCR. Phenotypic features of the genetrap allele recapitulate those of Prg4 knockout alleles (protein deposition on cartilage by 2 months of age, increased articular joint friction, etc. - see Stock No. 025737).

Cre-mediated excision of the genetrap restores lubricin expression and the wildtype condition. Scattered areas of β-galactosidase expression may still be present in the synovium of treated mice, however, suggesting that lubricin expression may not be restored in every type B synoviocyte.

Restoring gene function prior to conception through crosses with germline-specific EIIa-cre (see Stock No. 003724) prevents cartilage damage. Tamoxifen-induced restoration of gene function (through crosses with Gt(ROSA)26Sor^CreERt2 (see Stock No. 008463)) at 3-weeks-of-age improves, but does not normalize, joint histology or whole joint friction. Restoring gene function in 2-month-old or 6-month-old mice has no beneficial effect.

The gene trap design additionally includes a reverse tet transactivator (rtTA) cassette.

Development

A targeting vector was designed to contain a loxP-flanked artificial exon that contains a strong splice acceptor, an in-frame lacZ coding sequence, an internal ribosome entry site (IRES), a reverse tet transactivator (rtTA) cassette, and a neomycin selection cassette driven by its own promoter. The mutation was introduced to intron 2 of the Prg4 gene via homologous recombination in (129X1/SvJ x 129S1/Sv)F1- Kitl⁺-derived R1 embryonic stem (ES) cells. The donating laboratory maintained this strain on a mixed C57BL/6J-129 genetic background.

Allele

Symbol: Prg4^tm2Mawa
Name: targeted mutation 2, Matthew Warman
MGI accession ID: MGI:5581510
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed); Reporter; Null/Knockout; Inserted expressed sequence; Transactivator
Marker symbol: Prg4
Marker name: proteoglycan 4 (megakaryocyte stimulating factor, articular superficial zone protein)
Chromosome: 1
Strain of origin: (129X1/SvJ x 129S1/Sv)F1-Kitl⁺
Expressed genes: rtTA,reverse tetracycline-controlled transactivator,E. coli; Bgeo,fusion of beta-galactosidase and neomycin phosphotransferase genes,E. coli
Site of expression: ß-galactosidase instead of lubricin is expressed in cartilage.
Molecular note: A targeting vector containing a loxP-flanked artifical exon that contains a strong splice acceptor, an in-frame Bgeo lacZ coding sequence, an internal ribosome entry site (IRES), a reverse tet transactivator (rtTA) cassette with a polyA signal, and a neomycin selection cassette driven by its own promoter was inserted into intron 2 of the gene. The allele does not express wild-type Prg4 mRNA, as determined by RT-PCR. Cre-mediated excision of the targeting vector leaves one loxP in intron 2 and allows expression of Prg4 mRNA.