STOCK Dnmt1^tm3Jae Hprt^b-m3 Tg(pPWL512hyg)1Ems/J

Stock number: 003208
Product name: DR4
Strain synonyms: DR-4
Research areas: Metabolism Research, Neurobiology Research, Research Tools

Description

DR-4 transgenic mice harbor three independently segregating loci that collectively express four drug resistance genes, and thus provide a good source of multiple-drug resistant mouse embryonic fibroblasts.

Detailed description

These DR-4 mice harbor three independently segregating loci that collectively express four drug resistance genes. The Dnmt1^tm3Jae targeted mutation on chromosome 9 confers resistance to both neomycin/G418 and puromycin (while retaining Dnmt1 function). The pPWL512hyg transgene (unknown genomic insertion) results in hygromycin resistance. These three resistance genes are controlled by the promoter and polyadenylation signal of the murine 3-phosphoglycerate kinase gene (Pgk1), which ensures high levels of expression in both embryonic stem (ES) cells and mouse embryonic fibroblasts (MEFs). The Hprt^b-m3 mutation on the X chromosome confers resistance to 6-thioguanine (and abolishes endogenous Hprt function).

MEFs prepared from DR-4 mice display resistance to concentrations of the drugs G418, puromycin, 6-thioguanine and hygromycin well above those used normally for the selection of drug-resistant ES cells. This mouse strain represents an economical donor for the production of multiple-drug resistant MEFs.

Development

These DR-4 mice harbor a targeted mutation, a spontaneous mutation, and a transgene that collectively express four drug resistance genes. The Dnmt1^tm3Jae targeted mutation confers resistance to both neomycin/G418 and puromycin (while retaining Dnmt1 function). The Hprt^b-m3 spontaneous mutation confers resistance to 6-thioguanine (and abolishes endogenous Hprt function). Expression of the pPWL512hyg transgene results in hygromycin resistance. Each are described in more detail below.

The Dnmt1^tm3Jae mutation (chip) was created by Dr. Rudolf Jaenisch to have (from 5' to 3') the endogenous Dnmt1 promoter region, a wildtype Dnmt1 cDNA fragment, a PGK-puro^R-polyA cassette and a PGK-neo^R-polyA cassette. This was created by two targeting events. First, the pMT(S)neo targeting vector was designed to insert a PGK-neo^R-polyA cassette (murine 3-phosphoglycerate kinase promoter [Pgk1], neomycin resistance gene, and polyadenylation signal sequence [all in the same transcriptional orientation as the targeted gene]) into the unique SalI site of an exon in the 5' region of the Dnmt1 gene. This vector was electroporated into 129S4/SvJae-derived J1 embryonic stem (ES) cells. Correctly targeted ES cells bearing the Dnmt^s mutation (Dnmt1^tm1Enl) were then re-targeted via electroporation of the pMWT-PURO targeting vector. This vector was designed with a 4.9 kbp wildtype Dnmt1 cDNA fragment (encoding the entire coding region) followed by a PGK-puro^R-polyA cassette (murine 3-phosphoglycerate kinase promoter [Pgk1], puromycin resistance gene, and polyadenylation signal sequence). The resulting ES cells shown to be resistant to both neomycin and puromycin (Dnmt1^tm3Jae) were used to make chimeric males, which were bred with BALB/c females. Homozygous mice on a mixed BALB/c;129S4/SvJae genetic background were used as below.

The Hprt^b-m3 mutation is a deletion spanning the promoter and first two exons of the Hprt gene on the X chromosome. This mutation spontaneously arose in E14TG2a ES cells (originally isolated as a spontaneous Hprt-deficient, 6-thioguanine (6-TG)-resistant derivative of the 129P2/OlaHsd-derived E14 ES cell line). E14TG2a ES cells were used to generate mutant mice. Male chimeras were bred to C57BL/6 females. Female progeny were bred to 129/SvJae males, and the resulting offspring were bred together to generate homozygous animals. Homozygous mice on a mixed C57BL/6c;129/SvJae genetic background were used as below.

The pPWL512hyg transgene was created by Dr. Elizabeth M. Simpson (while at The Jackson Laboratory) with a 507 bp segment of the murine 3-phosphoglycerate kinase promoter (Pgk1), an optimized hygromycin B resistance gene (hyg^R; modified with optimal Kozak mammalian translation initiation signal), and a 452 bp polyadenylation signal sequence. The ~2 kbp transgene was microinjected into the male pronucleus of C57BL/6J embryos. One founder male with a single copy transgene insertion was bred to C57BL/6J females. The resulting C57BL/6J-TgN(pPWL5I2hyg)IEms mice were bred together for several generations as Stock No. 002354. Transgenic mice were used as below.

To generate DR-4 animals, C57BL/6c;129/SvJae-Hprt^b-m3 mice were bred with C57BL/6J-TgN(pPWL5I2hyg)IEms homozygotes. The resulting F1 progeny were bred with BALB/c;129S4/SvJae-Dnmt1^tm3Jae homozygotes. The resulting offspring were bred together to create the homozygous DR-4 strain. Homozygous mice were sent to The Jackson Laboratory Repository as Stock No. 003208.

Allele

Symbol: Tg(pPWL512hyg)1Ems
Name: transgene insertion 1, Elizabeth M Simpson
MGI accession ID: MGI:3052969
Generation method: Transgenic
Attributes: Inserted expressed sequence
Marker symbol: Tg(pPWL512hyg)1Ems
Marker name: transgene insertion 1, Elizabeth M Simpson
Chromosome: UN
Strain of origin: C57BL/6J
Expressed genes: hyg,hygromycin resistance,bacteria
Molecular note: The transgene contains a PGK promoter and the bacterial hygromycin resistance gene.
General note: Transgene also called "feeder" and "hyg."

Transgenic mice on a C57BL/6 background express the hygromycin resistance gene in a wide variety of tissues including muscle. Embryonic feeder layers derived from these mice are resistant to up to 0.8mg/ml hygromycin B, whereas feeder layers from normal C57BL/6J mice are killed by 0.2mg/ml hygromycin B.

Mouse embryonic fibroblasts prepared from homozygous transgenic mice that are also homozygous for Tg(Hprt1)1Jae and Dnmt1^tm3Jae are resistant to concentrations of the drugs G418, 6TG, puromycin, and hygromycin well above those used normally for the selection of drug-resistant ES cells.

Allele

Symbol: Dnmt1^tm3Jae
Name: targeted mutation 3, Rudolf Jaenisch
MGI accession ID: MGI:2657151
Generation method: Targeted
Attributes: Inserted expressed sequence
Marker symbol: Dnmt1
Marker name: DNA methyltransferase (cytosine-5) 1
Chromosome: 9
Strain of origin: 129S4/SvJae
Molecular note: A targeting vector containing a Dnmt1 cDNA fragment followed by a PGK-puro cassette was inserted upstream of the previously targeted coding region containing a neomycin selection cassette (Dnmt1^tm1Enl) and downstream of the endogenous promoter. Northern blot analysis showed that transcript produced by the inserted cDNA sequence was at a level and size similar to that of the endogenous allele.
General note: Previously targeted J1 ES cells homozygous for Dnmt1^tm1Enl were electroporated with the targeting vector.

Allele

Symbol: Hprt1^b-m3
Name: hypoxanthine guanine phosphoribosyl transferase B, mutation 3
MGI accession ID: MGI:1857299
Generation method: Spontaneous
Marker symbol: Hprt1
Marker name: hypoxanthine phosphoribosyltransferase 1
Chromosome: X
Strain of origin: 129P2/OlaHsd
Molecular note: The allele contains a ~55 kb deletion spanning the promoter and first 2 exons. Subsequent direct sequence comparison with wild type DNA defined the exact breakpoints of the deletion, which lies 415 bp after the 3' end of exon 2, and determined the deletion size to be 36 kb.
General note: HPRT- embryonic stem cells were obtained by selecting for spontaneous mutation by incubation in medium containing 6-thioguanine. HPRT- males have no overt phenotype of abnormal behavior (J:15483). The mutation is due to a large deletion in the Hprt gene. In situ hybridization studies showed HPRT mRNA in high levels in most neurons, but not in glial cells, in normal mice. No HPRT mRNA was detected in the brains of male mice carrying this deletion (J:2058). Mutant mice have no HPRT detectable by Western blot analysis and no detectable HPRT enzyme activity in brain homogenates. They appear to have normal brain purine content, but de novo purine synthesis is accelerated four- to fivefold (J:11842). The Hprt^b-m3 mutation has been used in preimplantation studies to determine when the maternal and paternal alleles of Hprt are activated during early embryonic development (J:2389). Either administration of amphetamine (J:1847) or inhibition of adenine phosphoribosyltransferase (APRT) activity (J:4123) stimulates locomotor and stereotypic behaviors in HPRT-deficient mice. However, the null mutant for both Hprt and Aprt does not show the characteristics of Lesch-Nyhan disease (J:35822). Cells from mice hemizygous or homozygous for this mutation are HPRT-deficient and resistant to the drug 6-thioguanine (6TG).