| Primary Identifier | MGI:4442339 | Allele Type | Targeted |
| Attribute String | Inducible, Inserted expressed sequence | Gene | Col1a1 |
| Transmission | Germline | Strain of Origin | (C57BL/6 x 129S4/SvJae)F1 |
| Induced With | doxycycline/tetracycline | Is Recombinase | false |
| Is Wild Type | false |
| description | The allele was made in KH2 cells that carry Gt(ROSA)26Sortm1(rtTA*M2)Jae and Col1a1tm13(neo/hygro*)Jae. This allele was generated with Col1a1tm13(neo/hygro*)Jae. |
| molecularNote | A targeting vector was designed to insert frt-flanked PGK-Neo-pA into the 3' UTR of the Col1a1 locus. This construct was electroporated into (C57BL/6 x 129S4Sv/Jae)F1-derived V6.5 embryonic stem (ES) cells. Correctly targeted ES cells were identified. One of these ES cells clones, C10, was retargeted to insert an optimized form of reverse tetracycline controlled transactivator (rtTA-M2) followed by a beta-globin intron and polyA signal downstream of the Gt(ROSA)26Sor promoter. Correctly targeted ES cells were identified. One of these ES cell clones, KH2, was selected and the frt-flanked PGK-Neo-pA in the 3' UTR of the Col1a1 locus was replaced by co-injection of a tetOP-OKSM "flip-in plasmid" and a CAG-Flpe plasmid. The tetOP-OKSM "flip-in plasmid" contained a splice acceptor-double polyA sequence and the tetracycline responsive element (TRE, tetOP, or tetO) upstream of the four mouse reprogramming genes Oct4 (Pou5f1; POU domain, class 5, transcription factor 1), Klf4 (Kruppel-like factor 4 (gut)), Sox2 (SRY-box containing gene 2), and c-Myc (Myc; myelocytomatosis oncogene) separated by three different sequences that mediate ribosomal skipping (F2A [from foot-and-mouth disease virus], IRES [internal ribosome entry site], and E2A [from equine rhinitis A virus], respectively). The resulting ES cells, now targeted with rtTA-M2 in the Gt(ROSA)26Sor locus (Rosa26-rtTA) and tetOP-OKSM in the Col1a1 locus (Col1a1-tetO-OKSM), were injected into recipient blastocysts. Chimeric mice were bred together to establish the double mutant colony. Double mutant mice were bred together, and some of the animals were chosen with the Col1a1-tetO-OKSM mutation and without the Rosa26-rtTA mutation. |
