Biotechnology Bulletin ›› 2023, Vol. 39 ›› Issue (2): 283-291.doi: 10.13560/j.cnki.biotech.bull.1985.2022-0863
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CHENG Jing-wen1(), CAO Lei1, ZHANG Yan-min1, YE Qian1, CHEN Min2, TAN Wen-song1, ZHAO Liang1,2()
Received:
2022-07-14
Online:
2023-02-26
Published:
2023-03-07
CHENG Jing-wen, CAO Lei, ZHANG Yan-min, YE Qian, CHEN Min, TAN Wen-song, ZHAO Liang. Establishment and Application of Multigene Engineering Transformation Strategy for CHO Cells[J]. Biotechnology Bulletin, 2023, 39(2): 283-291.
寡核苷酸链名称Oligos name | DNA序列DNA sequences(5'-3') |
---|---|
FUT8-sg1F | CACCGATGGACTGGTTCCTGGCGT |
FUT8-sg1 R | AAACACGCCAGGAACCAGTCCATC |
FUT8-sg2 F | CACCGTAAAACAATAAGGTCCCCC |
FUT8-sg2 R | AAACGGGGGACCTTATTGTTTTAC |
FUT8-sg3 F | CACCGAGAAGGCCCTATTGATCAG |
FUT8-sg3 R | AAACCTGATCAATAGGGCCTTCTC |
Table 1 sgRNA oligos
寡核苷酸链名称Oligos name | DNA序列DNA sequences(5'-3') |
---|---|
FUT8-sg1F | CACCGATGGACTGGTTCCTGGCGT |
FUT8-sg1 R | AAACACGCCAGGAACCAGTCCATC |
FUT8-sg2 F | CACCGTAAAACAATAAGGTCCCCC |
FUT8-sg2 R | AAACGGGGGACCTTATTGTTTTAC |
FUT8-sg3 F | CACCGAGAAGGCCCTATTGATCAG |
FUT8-sg3 R | AAACCTGATCAATAGGGCCTTCTC |
引物名称Primer name | DNA序列DNA sequence(5'-3') |
---|---|
FUT8-Exon1-F | AGAGTCATCACAGTATACCAGAGAG |
FUT8-Exon1-R | GCCACTGCTTCTATATACTGATTCA |
FUT8-Exon2-F | CATTCTCAGCTAGCCCTTATGATTA |
FUT8-Exon2-R | TATGGAAGCCCAAATGAAGCACA |
Table 2 PCR primers for T7E I enzymatic digestion assay
引物名称Primer name | DNA序列DNA sequence(5'-3') |
---|---|
FUT8-Exon1-F | AGAGTCATCACAGTATACCAGAGAG |
FUT8-Exon1-R | GCCACTGCTTCTATATACTGATTCA |
FUT8-Exon2-F | CATTCTCAGCTAGCCCTTATGATTA |
FUT8-Exon2-R | TATGGAAGCCCAAATGAAGCACA |
目的 Purpose | 引物名称 Primer name | DNA序列 DNA sequence(5'-3') |
---|---|---|
5' junction PCR | FUT8 5' F | AACTCTGATTTTTGGAATCCCCTTTCTTCAGC |
FUT8 5' R | TGGGTCTCCCTATAGTGAGTCGTATTAATTTCG | |
3' junction PCR | FUT8 3' F | ATGAAGCAGCACGACTTCTTCAAGTCC |
FUT8 3' R | GCAATGGATGCAAACAGTGGTGTGG | |
Out-out PCR | FUT8 5' F | AACTCTGATTTTTGGAATCCCCTTTCTTCAGC |
FUT8 3' R | GCAATGGATGCAAACAGTGGTGTGG |
Table 3 Primers for site-specific integration
目的 Purpose | 引物名称 Primer name | DNA序列 DNA sequence(5'-3') |
---|---|---|
5' junction PCR | FUT8 5' F | AACTCTGATTTTTGGAATCCCCTTTCTTCAGC |
FUT8 5' R | TGGGTCTCCCTATAGTGAGTCGTATTAATTTCG | |
3' junction PCR | FUT8 3' F | ATGAAGCAGCACGACTTCTTCAAGTCC |
FUT8 3' R | GCAATGGATGCAAACAGTGGTGTGG | |
Out-out PCR | FUT8 5' F | AACTCTGATTTTTGGAATCCCCTTTCTTCAGC |
FUT8 3' R | GCAATGGATGCAAACAGTGGTGTGG |
引物名称Primer name | DNA序列DNA sequence(5'-3') |
---|---|
M-Bcl2 F | TCACAGAAGGACAAGGTGGATT |
M-Bcl2 R | AATGCTGACCTGAGCTGGTTT |
H-Bcl2 F | GAACTGGGGGAGGATTGTGG |
H-Bcl2 R | CATCCCAGCCTCCGTTATCC |
GAPDH F | CATGGCCTTCCGTGTTCCTA |
GAPDH R | TGAAGTCGCAGGAGACAACC |
FUT8 F | GACCACCCTGACCATTCTAGC |
FUT8 R | CACGGACTCTTCCTGTAGCTG |
Table 4 Primers for qPCR
引物名称Primer name | DNA序列DNA sequence(5'-3') |
---|---|
M-Bcl2 F | TCACAGAAGGACAAGGTGGATT |
M-Bcl2 R | AATGCTGACCTGAGCTGGTTT |
H-Bcl2 F | GAACTGGGGGAGGATTGTGG |
H-Bcl2 R | CATCCCAGCCTCCGTTATCC |
GAPDH F | CATGGCCTTCCGTGTTCCTA |
GAPDH R | TGAAGTCGCAGGAGACAACC |
FUT8 F | GACCACCCTGACCATTCTAGC |
FUT8 R | CACGGACTCTTCCTGTAGCTG |
Fig. 2 Results of sgRNA gene editing efficiency M: DL2000 DNA marker. 1: Wild-type cells exon1 PCR amplification products. 2: Wild-type cells exon 2 PCR amplification products. 3: PCR amplification products of sgRNA1/Cas9 transfected cells. 4: Exon1-sgRNA2/Cas9 transfected cells PCR amplification products. 5: PCR amplification products of sgRNA3/Cas9 transfected cells
Fig. 4 Molecular identification results of the site-specific synchronous double-knock cell A: Electrophoresis results of 5' junction PCR. B: Electrophoresis results of 5' junction PCR. C: Electrophoresis results of out-out PCR. D: Sequencing results of 5'/3' junction PCR amplification products. M: DL5000 DNA marker. 1: Wildtype CHO-K1 cells. 2: Site-specific synchronous double-knock cell
Fig. 5 Gene expressions of the site-specific synchronous double-knock cell A: Relative mRNA expression of endogenous Bcl-2. B: Relative mRNA expression of exogenous Bcl-2. C: Relative mRNA expression of FUT8. D: EGFP protein expression. E: Bcl-2 protein expression. F: FUT8 protease function. ** P<0.01,**** P<0.000 1,n=3. The same below
Fig. 8 Evaluation on the anti-apoptotic properties of the site-specific synchronous double-knock cell A: Proportion of necrotic cells. B: Proportion of early apoptotic. C: Proportion of late apoptotic. D: Proportion of living cells. n=3
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