Pik3ca-flH1047R-T2A-EYFP-NLS
| Status | Available to order |
| EMMA ID | EM:15710 |
| Citation information | RRID:IMSR_EM:15710 Research Resource Identifiers (RRID) are persistent unique ID numbers assigned to help researchers cite key resources (e.g. antibodies, model organisms and software projects) in the biomedical literature to improve transparency and reproducibility in research. See https://www.rrids.org/ for more information. |
| International strain name | Pik3ca-flH1047R-T2A-EYFP-NLS |
| Alternative name | Pik3ca-flH1047R-T2A-EYFP-NLS |
| Strain type | Targeted Mutant Strains |
| Allele/Transgene symbol | Pik3ca, Unknown at present |
| Gene/Transgene symbol | Pik3ca, Unknown at present |
Information from provider
| Provider | Philip Jones |
| Provider affiliation | Pre Cancer, Welcome Sanger Institute |
| Additional owner | Taconic made the mouse for us as a service following our design and Taconic have no ownership of the strain. |
| Genetic information | Conditional floxed allele Pik3a H1047R mutant with C terminal T2A peptide -EYFP- nuclear localisation sequence reporter targeted to Pik3ca locus. In the targeting vector, exon 20 (including the splice acceptor site of intron 19), the endogenous STOP sequence and the 3’UTR region of the wild-type Pik3ca gene were flanked by loxP sites. A second Pik3ca exon 20 including the Pik3caH1047R mutation was introduced 3’ of the distal loxP site. Between the last amino acid and the translation termination codon of the duplicated exon 20 the following sequences were inserted: a self-cleaving T2A peptide, enhanced yellow fluorescent protein (EYFP) fused to a Nuclear Localization Signal (NLS), a STOP cassette and the 3′UTR region of the Pik3ca gene. Two positive selection markers were also introduced. A Neomycin resistance gene flanked by Frt sites was inserted 3′ of the 5′ loxP site before exon 20. A Puromycin resistance gene flanked by F3 sites was placed downstream of the 3′UTR from the duplicated region. An additional polyadenylation signal (hGHpA: human Growth Hormone polyadenylation signal) was inserted between the 3’ UTR and the distal loxP site to prevent downstream transcription of the mutated Pik3caH1047R exon 20 before Cre recombination. Finally, the vector included a distal thymidine kinase (Tk) gene at the 3’ end for negative selection. The targeting vector was generated using BAC clones from the C57BL/6J RPCIB-731 BAC library and transfected into the Taconic Biosciences C57BL/6NTac ES cell line. Homologous recombinant clones were isolated using double positive (NeoR and PuroR) and negative (Tk) selections. The appropriate insertion of the vector was assessed by PCR. The conditional knock-in allele was obtained after in vivo Flp-mediated removal of the selection markers. This allele expresses the wild-type p110α protein. The presence of the hGHpA cassette downstream of the wild-type exon 20 prevents transcription of the mutated H1047R exon 20 and the EYFP before Cre recombination. The constitutive knock-in allele is obtained after Cre-mediated deletion of wild-type exon 20 and the hGHpA, expresses a chimeric transcript harboring the mutated p110αH1047R protein fused to the T2A sequence and the EYFP open reading frame including the NLS. The expected co-translational cleavage at the T2A sequences result in co-expression of the mutated p110αH1047R and EYFP proteins under the control of the endogenous Pik3ca promoter. |
| Phenotypic information | Homozygous:NoneHeterozygous:None |
| Breeding history | Strain generated in C57BL/6 ES cells and maintained on pure C57BL/6 background since. |
| References |
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| Homozygous fertile | yes |
| Homozygous viable | yes |
| Homozygous matings required | no |
| Immunocompromised | no |
Information from EMMA
| Archiving centre | Mary Lyon Centre at MRC Harwell, Oxford, United Kingdom |
Literature references
- Organismal metabolism regulates the expansion of oncogenic PIK3CA mutant clones in normal esophagus.;Herms Albert, Colom Bartomeu, Piedrafita Gabriel, Kalogeropoulou Argyro, Banerjee Ujjwal, King Charlotte, Abby Emilie, Murai Kasumi, Caseda Irene, Fernandez-Antoran David, Ong Swee Hoe, Hall Michael W J, Bryant Christopher, Sood Roshan K, Fowler Joanna C, Pol Albert, Frezza Christian, Vanhaesebroeck Bart, Jones Philip H, ;2024;Nature genetics;56;2144-2157; 39169259