Table 1.
Primer sequence information for vector construction and RT-PCR
| 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 |