生物技术通报 ›› 2023, Vol. 39 ›› Issue (2): 283-291.doi: 10.13560/j.cnki.biotech.bull.1985.2022-0863
程静雯1(), 曹磊1, 张艳敏1, 叶倩1, 陈敏2, 谭文松1, 赵亮1,2()
收稿日期:
2022-07-14
出版日期:
2023-02-26
发布日期:
2023-03-07
作者简介:
程静雯,女,硕士,研究方向:动物细胞与组织工程;E-mail: CHENG Jing-wen1(), CAO Lei1, ZHANG Yan-min1, YE Qian1, CHEN Min2, TAN Wen-song1, ZHAO Liang1,2()
Received:
2022-07-14
Published:
2023-02-26
Online:
2023-03-07
摘要:
近年来,中国仓鼠卵巢(Chinese hamster ovary, CHO)细胞工程改造主要通过敲入或敲除基因来改变细胞某个单一功能,而敲入和敲除的基因往往无法在单次实验操作中同时发挥相应的功能,限制了多基因同步改造的应用。该研究选取细胞凋亡通路中抗凋亡蛋白即B淋巴细胞瘤-2(B-cell lymphoma-2, Bcl-2)基因为敲入基因、蛋白岩藻糖基化通路中岩藻糖合成关键酶即岩藻糖转移酶8(fucosyltransferase 8, FUT8)基因为敲除基因作为模型,利用CRISPR/Cas9技术建立定点同步敲入敲除基因编辑策略。利用该策略获得的单克隆细胞株Bcl-2蛋白过表达且FUT8蛋白酶功能缺失。经传代培养发现,由建立的定点同步敲入敲除基因编辑策略获得的细胞株在60 d内所编辑的基因表达稳定。相较于原野生型细胞,该细胞株表现出对血清剥夺的耐受度更高以及对死亡的抵抗能力更强。由此说明基于定点同步敲入敲除基因编辑策略具备一定可行性,可用于重组蛋白生产的CHO工程细胞株的构建。
程静雯, 曹磊, 张艳敏, 叶倩, 陈敏, 谭文松, 赵亮. CHO细胞多基因工程改造策略的建立及应用[J]. 生物技术通报, 2023, 39(2): 283-291.
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 |
表1 sgRNA寡核苷酸链
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 |
表2 T7E I酶切实验引物
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 |
表3 定点整合验证所需引物
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 |
表4 qPCR引物序列
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 |
图2 sgRNA基因编辑效率检测结果 M:DL2000 DNA分子量标准;1:野生型细胞外显子1处PCR产物;2:野生型细胞外显子2处PCR产物;3:转染sgRNA1/Cas9质粒的细胞PCR扩增产物;4:转染sgRNA2/Cas9质粒的细胞PCR扩增产物;5:转染sgRNA3/Cas9质粒的细胞PCR扩增产物
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
图4 定点同步双敲细胞分子鉴定结果 A:5'junction PCR电泳结果;B:3'junction PCR电泳结果;C:out-out PCR电泳结果;D:5'/3' junction PCR 扩增产物测序结果。M:DL5000 DNA分子量标准;1:野生型CHO-K1细胞;2:定点同步双敲细胞株
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
图5 定点同步双敲细胞基因表达水平 A:内源性Bcl-2基因相对mRNA表达水平;B:Bcl-2基因相对mRNA表达水平;C:FUT8基因相对mRNA表达水平;D:EGFP蛋白表达水平;E:Bcl-2蛋白表达水平;F:FUT8蛋白酶功能。** P<0.01,**** P<0.000 1,n=3,下同
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
图6 基因表达稳定性结果 A:细胞EGFP蛋白表达比例;B:细胞荧光平均强度
Fig. 6 Stability results of gene expression A: Proportion of EGFP protein expression in cells. B: Fluorescence mean intensity of cells
图7 无血清条件下细胞活率 图左:72 h细胞活率;图右:96 h细胞活率。* P<0.05,n=3。下同
Fig. 7 Cell viability under serum-free condition Left part: Cell viability at 72 h. Right part: Cell viability at 96 h. * P<0.05,n=3. The same below
图8 定点同步双敲细胞抗凋亡性能评估 A:坏死细胞比例;B:早期凋亡细胞比例;C:晚期凋亡细胞比例;D:活细胞比例。n=3
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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