Table 1 Primer sequence information for vector construction and RT-PCR
From: Generation of an mESC model with a human hemophilia B nonsense mutation via CRISPR/Cas9 technology
Target | Forward/reverse primer (5′–3′) | |
|---|---|---|
House-keeping genes (RT-qPCR) | Gapdh | AACTTTGGCATTGTGGAAGGGCTCA/ TTGGCAGCACCAGTGGATGCAGGGA |
Pluripotency marker (RT-qPCR) | Nanog | CTCAAGTCCTGAGGCTGACA/ TGAAACCTGTCCTTGAGTGC |
Pluripotency marker (RT-qPCR) | Oct4 | TAGGTGAGCCGTCTTTCCAC/ GCTTAGCCAGGTTCGAGGAT |
Pluripotency marker (RT-qPCR) | Sox2 | CTGCAGTACAACTCCATGACCAG/ GGACTTGACCACAGAGCCCAT |
Pluripotency marker (RT-qPCR) | Klf4 | AACATGCCCGGACTTACAAA/ TTCAAGGGAATCCTGGTCTTC |
Endoderm marker (RT-qPCR) | Sox17 | CGAGCCAAAGCGGAGTCTC/ TGCCAAGGTCAACGCCTTC |
Endoderm marker (RT-qPCR) | Gata4 | CCCTACCCAGCCTACATGG/ ACATATCGAGATTGGGGTGTCT |
Mesoderm marker (RT-qPCR) | T | CATCGGAACAGCTCTCCAACCTAT/ GTGGGCTGGCGTTATGACTCA |
Mesoderm marker (RT-qPCR) | MixL1 | ACGCAGTGCTTTCCAAACC/ CCCGCAAGTGGATGTCTGG |
Ectoderm marker (RT-qPCR) | Pax6 | GCAGATGCAAAAGTCCAGGTG/ CAGGTTGCGAAGAACTCTGTTT |
Ectoderm marker (RT-qPCR) | Nestin | CCCTGAAGTCGAGGAGCTG/ CTGCTGCACCTCTAAGCGA |
Define endoderm marker (RT-qPCR) | Hnf-4α | GCCAACGATCACCAAGCAAG/ GCCAAAAGGCTCACACCAAAG |
Hepatoblast marker (RT-qPCR) | Afp | CGTGATGCTTTGGGCGTTTA/ GCCAAAAGGCTCACACCAAAG |
Hepatocyte-like cell marker (RT-qPCR) | Ttr | CCCTGCTCAGCCCATACTCCTA/ TGCTTTGGCAAGATCCTGGT |
Hepatocyte-like cell marker (RT-qPCR) | Alb | AAACCTTGTCACTAGATGCAAAGACG/ GGGTAGCCTGAGAAGGTTGTGG |
Target gene (RT-qPCR) | F9 | TGGAAGCAGTATGTTGATGGAGA/ CCAACTTGGCACCAGCATTC |
Guide RNA (px330) | Intron 2 | CACCGAAAGGAGTGTGGAACTAGAG/ AAACCTCTAGTTCCACACTCCTTTC |
Blunt-sequencing (PCR) | Left arm (1176 bp) | CATGAATTTCAGTGAAATGCTTCCATCACTGGC/ CCTGTGTATGTACTACTCAAAAAGGTCAGAAGAGAGTG |
Blunt-sequencing (PCR) | Blunt-Left arm- Mutation (3032) | GAAGAAAGATGTAGTTTTGAAGAAGCATGAGAAG/ CTTCTCATGCTTCTTCAAAACTACATCTTTCTTC |
Blunt-sequencing (PCR) | PGK-puro (1145 bp) | CCCAAGTAATAGGGGTAGGGGAGGCGCTTTTCCCAAGGCAGTCTG/ GAAGTTAGTGGCGGCACCGG GCTTGCGGGTCATG |
Blunt-sequencing (PCR) | P2A-mcherry (988 bp) | CAAGCCCGGTGCCGCCACTAACTTCAGCTTGTTGAAGCAG/ GGTCAGGGTGCAGGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTG |
Donor construction (PCR) | PGGA-Left arm(842 bp) | GGCTACGGTCTCTGGAGCAAACAGGCTTCTGTCCTTC/ GGCTACGGTCTCATACGAAGTTATCCTGTGTATGTACT ACTCAAAAAG |
Donor construction (PCR) | Loxp-PGK-puro (1161 bp) | GGCTACGGTCTCACGTATAATGTATGCTATACGAAGTTATGGGTAGGG/ GGCTACGGTCTCCTTTCCAGGAAGGCGGGCACCCC |
Donor construction (PCR) | mCherry-loxp (943 bp) | GGCTACGGTCTCCGAAACCTCCGCGCCCCGCAAC/ GGCTACGGTCTCGAACTTCGTATAGCATACATTATACG AAGTTATTTACT |
Donor construction (PCR) | PGGA-Right arm(758 bp) | GGCTACGGTCTCGAGTTATGTGCATGTATGCACACAGATAGAA/ GGCTACGGTCTCGATGGGACCAGAACACATGTGTCTAAAATGACAG |
F9-Flank | F9 | CATGAATTTCAGTGAAATGCTTCCATCACTGGC/ TGCTACTTCCATTTGTCACGTCCTGC |