B6.129(Cg)-Ptf1a^{tm2(cre/ESR1)Cvw} Kras^tm4Tyj Pten^tm1Hwu/J

Stock number: 033964
Product name: Ptf1a^Cre-ERTM;Kras^LSL-G12D;Pten^flox (also called KPP)
Strain synonyms: KPP
Research areas: Cancer Research, Cell Biology Research

Description

Ptf1a^Cre-ERTM;Kras^LSL-G12D;Pten^flox is a tamoxifen-inducible triple mutant strain (also called KPP) that may be useful as a model of pancreatic adenocarcinoma (PDA)-induced cachexia - a wasting syndrome characterized by pronounced loss of skeletal and cardiac muscle, and adipose tissues.

Detailed description

Ptf1a^Cre-ERTM;Kras^LSL-G12D;Pten^flox is a triple mutant strain (also called KPP) that may be useful as a tamoxifen-inducible model of pancreatic adenocarcinoma (PDA)-induced cachexia - a wasting syndrome characterized by pronounced loss of skeletal and cardiac muscle, and adipose tissues [Talbert et al. 2019 Cell Rep. 28:1612].

Each mutant allele is described below.

The Ptf1a^{tm2(cre/ESR1)Cvw} [Ptf1a^Cre-ERTM] knock-in/knock-out allele abolishes pancreas specific transcription factor 1a (Ptf1a) gene function and also expresses a tamoxifen-inducible Cre recombinase fusion protein (CreERTM) from the endogenous Ptf1a promoter/enhancer elements.
Of note, the Ptf1a^{tm2(cre/ESR1)Cvw} donating investigator's laboratory reported a different Ptf1a-cre line exhibited some paternal germline Cre recombination, and to be cautious, they suggest that the Ptf1a^Cre-ERTM allele be passed on maternally to avoid/minimize the possibility of germline deletion of floxed allele(s) [see Stock No. 019378].
If the recombinase activity pattern of this Ptf1a^Cre-ERTM allele is characterized by The Jackson Laboratory, such findings will be reported on the Mouse Genome Informatics (MGI) Allele Detail entry. This same information may also be found searching the MGI Recombinase Activity and MGI Gene Expression + Recombinase Activity Comparison Matrix.

The Kras^tm4Tyj allele [Kras^LSL-G12D] contains a point mutation (G12D) whose expression is blocked by the presence of a loxP-flanked stop codon. Mice homozygous for this Kras^tm4Tyj allele die in utero. Cre-mediated recombination can excise the stop codon and permit the oncogenic protein to be expressed.

The Pten^tm1Hwu allele [Pten^flox] contains loxP sites flanking exon 5 of the phosphatase and tensin homolog gene. Cre-mediated deletion of the floxed sequence results in a knock-out allele.

Development

This Ptf1a^Cre-ERTM;Kras^LSL-G12D;Pten^flox triple mutant line was generated by crossing three single mutant strains:
C57BL/6J-congenic Ptf1a^{tm2(cre/ESR1)Cvw}/J mice from Stock No. 019378,
B6.129S4-Kras^tm4Tyj/J mice from Stock No. 008179,
B6.129S4-Pten^tm1Hwu/J mice from Stock No. 006440.

For the Ptf1a^{tm2(cre/ESR1)Cvw} allele [Ptf1a^Cre-ERTM], targeting vector was designed to replace exons 1-2 of the pancreas specific transcription factor, 1a gene (Ptf1a) with a "loxed cassette acceptor" (LCA) to generate Ptf1a^LCA. The LCA contained a reverse-oriented neomycin resistance cassette (neo^R) that was flanked by inward-facing loxP sites. Next, a Ptf1a^{CreERTMHygroR} cassette exchange vector was constructed by inserting a 4105 bp fragment of Ptf1a into a plasmid containing two inversely oriented loxP sites, and a 5' UTR NotI site was changed to FseI. DNA encoding Cre-ERTM (Cre-ER^T2; Cre recombinase fused to a G525R mutant form of the mouse estrogen receptor ligand binding domain) was then inserted between this FseI and a natural MluI site in Ptf1a exon 1. A pgk-driven hygromycin resistance gene (HygroR), flanked by tandem frt sites, was inserted at the 3' end of the exchange vector for positive selection during recombinase-mediated cassette exchange (RMCE). The Ptf1a^{CreERTMHygroR} cassette exchange vector (along with a cre-expression plasmid to facilitate insertion of the exchange construct) was electroporated into 129S6/SvEvTac-derived TL-1 embryonic stem (ES) cells containing the Ptf1a^LCA allele. ES cells, resistant to both neo and hygro, were injected into blastocysts and resulting chimeric mice were bred to C57BL/6J to establish a colony. These mice were subsequently bred to Flp transgenic mice to delete the HygroR cassette, and progeny were crossed to remove the Flp-expressing transgene. The donating investigator reported that these Ptf1a^Cre-ERTM mice are maintained on a mixed background. Upon arrival, sperm was cryopreserved. To establish our live colony, an aliquot of frozen sperm was used to fertilize C57BL/6J oocytes (Stock No. 000664). The mice were then backcrossed to C57BL/6J inbred mice (Stock No. 000664) for five generations.

For the Kras^tm4Tyj allele [Kras^LSL-G12D], a targeting vector was designed to place a G12D point mutation in exon 1 of the gene and a loxP-flanked STOP element upstream of the mutation. The STOP element incorporates a PGK-puromycin selection cassette at the 5' end in an opposite directional orientation. An adenoviral strong splice acceptor, typically used in gene trap vectors, is fused upstream of the his3 stuffer fragment to prevent splicing around the stopper (in case the transcription isn't completely silenced). A mutant splice donor site is on the 3' end and as well as a tetrameric tandem array of SV40 PolyA. The stopper was designed to fit into genomic Sal1 or Xho1 sites. The stop cassette prevents the expression of mutant Kras until it is removed by Cre mediated recombination of the loxP sites, thus allowing expression of the oncogenic Kras gene. The construct was electroporated into 129S4/SvJae-derived J1 embryonic stem (ES) cells. This strain was crossed to 129S4/SvJae for more than 10 generations by the donating laboratory which verified correct orientation by Southern assays involving 5' and 3' external and internal probes. The Kras^LSL-G12D mice were then backcrossed onto the C57BL/6J background for 14 generations.

For the Pten^tm1Hwu allele [Pten^flox], a loxP site flanked targeting vector containing hygromycin resistance and thymidine kinase genes was used in the construction of this mutant. This selection cassette was inserted downstream of exon 5 of the targeted gene, and another loxP site was inserted upstream of exon 5. This construct was electroporated into 129S4/SvJae derived LW-1 embryonic stem (ES) cells, which were transiently transfected with a Cre-recombinase vector to remove the selection cassette. Correctly targeted ES cells were injected into BALB/c blastocysts. The resulting chimeric animals were crossed to BALB/cAnNTac mice before being made homozygous. The Pten^floxmice were then backcrossed onto the C57BL/6J background for 12 generations.

Allele

Symbol: Ptf1a^{tm2(cre/ESR1)Cvw}
Name: targeted mutation 2, Christopher VE Wright
MGI accession ID: MGI:3771322
Generation method: Targeted
Attributes: Recombinase-expressing; Inducible
Marker symbol: Ptf1a
Marker name: pancreas specific transcription factor, 1a
Chromosome: 2
Strain of origin: 129S6/SvEvTac
Expressed genes: cre/Esr1,Cre recombinase and estrogen receptor 1 fusion gene,
Site of expression: The endogenous Ptf1a promoter/enhancer elements direct expression of tamoxifen-inducible Cre recombinase fusion protein (CreERTM) to pancreatic acinar cells.
Molecular note: RMCE using Ptf1a^tm1Mgn inserted a cre and mouse Esr1 (CreERTM) replacing exon 1 and 2 and a hygromycin resistance cassette downstream of exon 2. Flp-mediated recombination removed the FRT-flanked hygro cassette.

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
Marker symbol: Pten
Marker name: phosphatase and tensin homolog
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); No functional change
Marker symbol: Kras
Marker name: Kirsten rat sarcoma viral oncogene homolog
Chromosome: 6
Strain of origin: 129S4/SvJae
Site of expression: Cre-inducible expression of oncogenic Kras^G12D protein
Molecular note: By homologous recombination in ES cells, the Kras2 locus was targeted with a cassette containing an oncogenic form of the KRAS2 protein in which the glycine at position 12 had been substituted with a an aspartic acid. A loxP flanked stop codon was included upstream of the inserted Kras2 sequence, such that the mutant transcript would be expressed only after cre-mediated recombination.