STOCK Gt(ROSA)26Sor^{tm3(CAG-EYFP)Hze} Kras^tm4Tyj Nkx3-1^{tm4(cre/ERT2)Mms} Pten^tm1Hwu/AbshnJ

Stock number: 033761
Product name: NPK
Research areas: Cancer Research, Cell Biology Research, Research Tools

Description

These NPK mice contain the Rosa26-CAG-LSL-EYFP, Kras^LSL-G12D, Nkx3.1^CreERT2, and Pten^flox targeted mutations and serve as a good tool to study the progression of prostate cancer in a mouse model.

Detailed description

The Nkx3.1^CreERT2 allele is used to achieve inducible Cre recombination specifically in the prostatic epithelium. This allele contains a knock-in of a tamoxifen-inducible CreERT2 under the control of the endogenous NK3 homeobox 1 (Nkx3.1) promoter, which results in inactivation of the NKX3.1 gene. Heterozygous inactivation of NKX3.1 in the Nkx3.1^CreERT2/+ mice leads to a preinvasive phenotype in the prostatic epithelium. The Pten floxed allele has loxP sites flanking exon 5 of the phosphatase and tensin homolog (Pten) gene. In the Kras^LSL-G12D allele, Kras G12D mutant is expressed under the control of endogenous Kirsten rat sarcoma viral oncogene homolog (Kras) promoter and a loxP-flanked STOP element, which prevents expression of oncogenic KRAS until it is removed by Cre mediated recombination of the loxP sites. The Rosa26-CAG-LSL-EYFP allele contains EYFP inserted at the Rosa26 locus, which is activated by Cre-mediated recombination.

Induction of Cre by tamoxifen administration leads to gene recombination, resulting in inactivation of Pten, activation of Kras^G12D expression at endogenous levels, and EYFP lineage marking of prostatic epithelial cells.

The Nkx3.1^CreERT2/+ ; Pten^flox/flox ; Kras^LSL-G12D/+; Rosa26-CAG-LSL-EYFP/+ mice develop lethal prostate cancer, where tumors are mostly undifferentiated often combined with sarcomatoid areas. These mice also develop metastatic phenotypes with high penetrance of metastasis to bone.

Development

The Nkx3.1^CreERT2 targeting vector was designed to insert a cre/ERT2 fusion gene (Cre recombinase fused to a human estrogen receptor ligand binding domain) followed by a polyadenylation sequence into the translation start site of the NK3 homeobox 1 (Nkx3-1) gene. The construct also included a self-excising ACN cassette for positive selection and a PGK-tk (thymidine kinase) cassette for negative selection. The ACN cassette, containing the neomycin resistance gene and Cre recombinase gene under the control of angiotensin-converting enzyme promoter, is flanked by loxP sites. Cre-mediated recombination during spermatogenesis removes the cassette leaving one loxP site. The construct was electroporated into 129S6/SvEvTac-derived TC1 embryonic stem (ES) cells. Correctly targeted ES cells were injected into blastocysts and resulting chimeric mice were bred with C57BL/6 mice to establish a colony.

This new strain, Stock No. 033761, was generated by crossing the resulting offspring to Rosa26-CAG-LSL-EYFP mice (Stock No. 007903), to Pten^flox mice (Stock No. 006440), and subsequently to Kras^LSL-G12D mice (Stock No. 008179). Mice are maintained on a mixed genetic background that contains mostly C57BL/6.

Allele

Symbol: Nkx3-1^{tm4(cre/ERT2)Mms}
Name: targeted mutation 4, Michael M Shen
MGI accession ID: MGI:4365630
Generation method: Targeted
Attributes: Recombinase-expressing; Inducible; Null/Knockout
Synonyms: Nkx3-1^CreERT2
Marker symbol: Nkx3-1
Marker name: NK3 homeobox 1
Marker synonyms: bagpipe; BAPX2; Bax; NKX3; Nkx3.1; Nkx-3.1; NKX3A
Chromosome: 14
Strain of origin: 129S6/SvEvTac
Site of expression: Prostate.
Molecular note: A cre/ERT2-polyA sequence was inserted at the translation start site of the Nkx3-1 gene, thus generating a null allele. The targeting vector also contained a self-excising ACE-cre/PolII-neo selection cassette for positive selection and a PGK-tk (thymidine kinase) cassette for negative selection. The vector was transfected into TC-1 ES cells which were used to generate chimeras. Excision of the selection cassette occurred by passage through the male germline.

Allele

Symbol: Gt(ROSA)26Sor^{tm3(CAG-EYFP)Hze}
Name: targeted mutation 3, Hongkui Zeng
MGI accession ID: MGI:3809521
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed); Reporter
Synonyms: Ai3; R26^LSL-YFP; Rosa-CAG-LSL-EYFP-WPRE
Marker symbol: Gt(ROSA)26Sor
Marker name: gene trap ROSA 26, Philippe Soriano
Marker synonyms: beta geo; Gtrgeo26; Gtrosa26; R26; ROSA26; SETD5-AS1; Thumpd3as1
Chromosome: 6
Strain of origin: (129S6/SvEvTac x C57BL/6NCrl)F1
Expressed genes: YFP,Yellow Fluorescent Protein,jellyfish
Site of expression: A floxed STOP prevents transcription of EYFP until mice are bred to a Cre strain. Resulting offspring express EYFP in a pattern dictated by the Cre promoter.
Molecular note: The Rosa-CAG-LSL-EYFP-WPRE targeting vector was designed with (from 5' to 3') a CMV-IE enhancer/chicken beta-actin/rabbit beta-globin hybrid promoter (CAG), an FRT site, a loxP-flanked STOP cassette (with stop codons in all 3 reading frames and a triple polyA signal), an enhanced yellow fluorescent protein sequence (from pcDNA6.2/N-YFP-DEST), a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE; to enhance the mRNA transcript stability), a polyA signal, and an attB/attP-flanked PGK-FRT-Neo-polyA cassette. This entire construct was inserted between exons 1 and 2 of the Gt(ROSA)26Sor locus.

Allele

Symbol: Pten^tm1Hwu
Name: targeted mutation 1, Hong Wu
MGI accession ID: MGI:2156086
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed); No functional change
Synonyms: Pten^ex5lox; PTEN^F; Pten^fl; Pten^flox; Pten^flx; Pten^fx; Pten^lox; Pten^loxp
Marker symbol: Pten
Marker name: phosphatase and tensin homolog
Marker synonyms: 10q23del; 2310035O07Rik; A130070J02Rik; B430203M17Rik; BZS; CWS1; DEC; GLM2; MHAM; Mmac; MMAC1; PTEN1; PTENbeta; PTENgama; TEP1
Chromosome: 19
Strain of origin: 129S4/SvJae
Site of expression: Pten is a widely expressed tumor suppresser gene.
Molecular note: A loxP flanked hygromycin resistance cassette was inserted 5' to exon 5, and a single loxP site was inserted 3' to exon 5, which encodes the phosphatase domain. The hygromycin cassette was removed in ES cells by transient Cre recombinase expression prior to the production of chimeric mice, leaving a single loxP site in place of the cassette. These insertions do not appear to have any effect on the normal function of the gene.
General note: Phenotypic Similarity to Human Syndrome: PTEN Hamartoma Tumor Syndrome (see OMIM:601728 Phosphatase And Tensin Homolog; PTEN) J:199362

Allele

Symbol: Kras^tm4Tyj
Name: targeted mutation 4, Tyler Jacks
MGI accession ID: MGI:2429948
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed); Null/Knockout
Synonyms: caKRas; KR; K-Ras(G12D)^fl; Kras^G12D; K-Ras^L; Kras^Lox; K-ras^LSL; Kras^LSL-G12D; Kras2^tm4Tyj; Kras2tm14Tyj; K-RasG12D; Lox-Stop-Lox-Kras^G12D; LSL-Kras G12D; LSL-K-ras G12D; LSL-Kras^G12D; LSL-K-ras^G12D; LSL-KrasG12D
Marker symbol: Kras
Marker name: Kirsten rat sarcoma viral oncogene homolog
Marker synonyms: CFC2; c-Ki-ras; c-Ki-ras2; 'C-K-RAS; C-K-RAS; Ki-ras; K-ras; K-Ras 2; KRAS1; KRAS2; Kras-2; K-RAS2A; K-RAS2B; K-RAS4A; K-RAS4B; NS; NS3; OES; p21; RALD; RALD2; RASK2
Chromosome: 6
Strain of origin: 129S4/SvJae
Site of expression: Cre-inducible expression of oncogenic Kras^G12D protein
Molecular note: Glycine codon 12 was changed to aspartic acid (p.G12D) to encode an oncogenic form of the KRAS2 protein. A loxP site flanked STOP cassette was included upstream of the inserted Kras2 sequence, such that the mutant transcript would be expressed only after Cre-mediated recombination.