STOCK Gt(ROSA)26Sor^{tm3(CAG-EYFP)Hze} Nkx3-1^{tm4(cre/ERT2)Mms} Pten^tm1Hwu Tg(ARR2/Pbsn-MYC)7Key/AbshnJ

Stock number: 033757
Product name: NPM
Research areas: Cancer Research, Cell Biology Research, Research Tools

Description

These NPM mice contain the Rosa26-CAG-LSL-EYFP, Nkx3.1^CreERT2, and Pten^flox targeted mutations, and the Hi-Myc transgene with over expression of flag-tagged human Myc in the prostate. These mice 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. The Pbsn-MYC transgene expresses the human myelocytomatosis oncogene (MYC) cDNA under the control of a truncated rat probasin (Pbsn) promoter with 2 additional androgen-inducible elements. 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 and EYFP lineage marking of prostatic epithelial cells. MYC is constitutively expressed in the prostatic epithelium beginning in puberty.

The Nkx3.1^CreERT2/+ ; Pten^flox/flox ; Tg-Pbsn-MYC ; Rosa26-CAG-LSL-EYFP/+ mice develop lethal prostate cancer with often mixed tumors combining adenocarcinoma, squamous carcinoma and sarcomatoid growth and metastatic phenotypes. Upon castration, these mice develop mostly sarcomatoid tumors with heterologous components.

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.

The Hi-Myc transgene was designed with nucleotides -426 through +28 of the rat probasin (Pbsn) gene, containing its androgen-inducible, prostate-specific promoter and 2 additional androgen-inducible elements, directing expression of a flag-tagged human MYC cDNA followed by a poly-adenylation signal. This transgene was microinjected into fertilized FVB oocytes. Hi-Myc mice from founder line 7 were maintained on an FVB/N genetic background.

This new strain, Stock No. 033757, was generated by crossing the resulting double allelic offspring to Rosa26-CAG-LSL-EYFP mice (Stock No. 007903) and subsequently to Pten^flox mice (Stock No. 006440). Mice are maintained on a mixed genetic background that contains mostly C57BL/6.

Allele

Symbol: Tg(ARR2/Pbsn-MYC)7Key
Name: transgene insertion 7, Katharine Ellwood-Yen
MGI accession ID: MGI:3785495
Generation method: Transgenic
Attributes: Inserted expressed sequence; Humanized sequence
Synonyms: ARR2/probasin-Myc; ARR2PB-Myc-PAI; Hi-Myc; Tg(Pbsn-MYC)4Key
Marker symbol: Tg(ARR2/Pbsn-MYC)7Key
Marker name: transgene insertion 7, Katharine Ellwood-Yen
Marker synonyms: ARR2PB-Myc-PAI; Hi-Myc; Tg(Pbsn-MYC)4Key
Chromosome: UN
Strain of origin: FVB
Site of expression: Prostate
Molecular note: Nucleotides -426 through +28 of the rat probasin gene, containing its androgen-inducible, prostate-specific promoter with 2 additional androgen-inducible elements, were joined to a human MYC cDNA flanked by a FLAG epitope coding sequence and the poly-adenylation signal from the insulin receptor gene. Immunoblot analysis with an antibody specific for human MYC demonstrates expression in prostate of mice as young as two weeks of age, which increases as the mice approach puberty at approximately 4 weeks. Four founder lines were established (4, 7, 11 and 13) since no distinction was made between the lines, line 7 was chosen as the representative line.

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