Table 1:
Primers for selection and characterization of Col11a1 targeted ES cells and conditional mice.
| Reaction name | Primer name | Col11a1 primer sequence | Size | Detect | Function |
|---|---|---|---|---|---|
| LacZ Insertion | LacZ FW LacZ RV |
ATCACGACGCGCTGTATC ACATCGGGCAAATAATATCG |
108 bp | targeted vector targeted allele |
Vector characterization ES cell selection |
| FRT | 5FRT_F | AGGCGCATAACGATACCACGAT | 204 bp | targeted vector | Vector characterization |
| 5FRT_R | CCACAACGGGTTCTTCTGTT | targeted allele | ES cell selection | ||
| DTA negative selection | DTA-5 DTA-3 |
AGGGAAGGCTGAGCACTACA CATTCTGCACGCTTCAAAAG |
506 bp | targeted vector | Vector characterization ES cell selection |
| 5 arm | 5 arm FW 5 arm RV |
TTCAGTGGCATGGTTTTCAA CCAACCCCTTCCTCCTACAT |
250 bp | targeted vector targeted allele |
ES cell selection targeted mice section |
| 3 arm | exon1 FW 3 arm RV |
TCAGCCAACTGAACAACGAC CATCCTCTTCTCCTGCCTTG |
166 bp 203 bp |
wild type, targeted, and floxed alleles | genotyping wild type, targeted, and floxed alleles |
| 5’ insertion/ orientation* | 5a FW 5b FW 5 RV |
CCATAGGTGATCACAAGAGTTTGC GAATAATGCCAGAGTGAACACACC CACAACGGGTTCTTCTGTTAGTCC |
6299 bp 5397 bp |
targeted allele targeted allele |
ES cell selection ES cell selection |
| 3’ insertion/ orientation* | 3 FW 3a RV 3c RV |
CACACCTCCCCCTGAACCTGAAAC CTTGGAATCAGGGAGGAGAAAAAG CACCCCAGTGACCACATCTTTAGC |
7736 bp 8312 bp |
targeted allele targeted allele |
ES cell selection ES cell selection |
| tm1c | tm1c_F tm1c_R |
AAGGCGCATAACGATACCAC CCGCCTACTGCGACTATAGAGA |
218 bp | floxed allele | floxed mice selection |
| Scx Cre | ra47 ra48 |
GCAGAACCTGAAGATGTTCGC ACACCAGAGACGGAAATCCATC |
500 bp | Cre transgene | Cre mice selection |
| Rosa Cre | wt forw wt rev mut rev |
AAAGTCGCTCTGAGTTGTTAT GGAGCGGGAGAAATGGATATG CCTGATCCTGGCAATTTCG |
825 bp 8312 bp |
Rosa Cre allele wild type |
TM-induced Cre mice genotyping |
| Cre excised | 5 arm FW 3 arm RV |
TTCAGTGGCATGGTTTTCAA CATCCTCTTCTCCTGCCTTG |
321 bp | Cre-excised allele | Cre-excision characterization |
*
PCR products are ~5–9kb. Long range PCR (LRPCR) will be performed (Invitrogen SequalPrep LRPCR kit) between a universal primer in the 5’ or 3’ arm of the targeting cassette and 2 different genomic specific primers in the region upstream or downstream of the construct.