Biotechnology Bulletin ›› 2021, Vol. 37 ›› Issue (5): 67-75.doi: 10.13560/j.cnki.biotech.bull.1985.2020-0963
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XIE Shao-yi(), JIANG Lu-man, YANG Xiao-feng, ZHANG Xian-yu, WU Zhen-fang, LI Zi-cong()
Received:
2020-08-03
Online:
2021-05-26
Published:
2021-06-11
Contact:
LI Zi-cong
E-mail:593293238@qq.com;lizicong@scau.edu.cn
XIE Shao-yi, JIANG Lu-man, YANG Xiao-feng, ZHANG Xian-yu, WU Zhen-fang, LI Zi-cong. Construction and Verification of CRISPR/Cas9 System Expression Vector for Mouse X Chromosome Cutting[J]. Biotechnology Bulletin, 2021, 37(5): 67-75.
引物名称 Primer name | 引物序列 Primer sequence(5'-3') | 产物长度 Product size/bp |
---|---|---|
X1-F | TGAGTACTTGGTTAGGGTGAGC | 1 212 |
X1-R | TGGAAGTCTCTGGCATA | |
X3-F | AAGGTTCCCAATGGTCACAGG | 1 383 |
X3-R | ACCATACCATGGTTTTCCCCA |
Table 1 Target fragment amplification primers
引物名称 Primer name | 引物序列 Primer sequence(5'-3') | 产物长度 Product size/bp |
---|---|---|
X1-F | TGAGTACTTGGTTAGGGTGAGC | 1 212 |
X1-R | TGGAAGTCTCTGGCATA | |
X3-F | AAGGTTCCCAATGGTCACAGG | 1 383 |
X3-R | ACCATACCATGGTTTTCCCCA |
引物名称 Primer name | 引物序列 Primer sequence(5'-3') | 产物长度 Product size/bp |
---|---|---|
sgRNA X1-F | TAATACGACTCACTATAGGGG- CTTGGTTAGGGTGAGTGCT | 120 |
sgRNA X1-R | AAAAGCACCGACTCGGTGCC | |
sgRNA X3-F | TAATACGACTCACTATAGGG- TAAGTGCTGTGTGCTGCTAC | 120 |
sgRNA X3-R | AAAAGCACCGACTCGGTGCC |
Table 2 Primers for amplification of sgRNA transcription template in vitro
引物名称 Primer name | 引物序列 Primer sequence(5'-3') | 产物长度 Product size/bp |
---|---|---|
sgRNA X1-F | TAATACGACTCACTATAGGGG- CTTGGTTAGGGTGAGTGCT | 120 |
sgRNA X1-R | AAAAGCACCGACTCGGTGCC | |
sgRNA X3-F | TAATACGACTCACTATAGGG- TAAGTGCTGTGTGCTGCTAC | 120 |
sgRNA X3-R | AAAAGCACCGACTCGGTGCC |
引物名称 Primer name | 引物序列 Primer sequence(5'-3') | 产物片段 Product size/bp |
---|---|---|
X1 mutation-F | GCATGCTTGGTTAGGGTGAGTT | 954 |
X1 mutation-R | GCTGGAAACCCAAGCAC |
Table 3 Primers for amplification of the X1 target site
引物名称 Primer name | 引物序列 Primer sequence(5'-3') | 产物片段 Product size/bp |
---|---|---|
X1 mutation-F | GCATGCTTGGTTAGGGTGAGTT | 954 |
X1 mutation-R | GCTGGAAACCCAAGCAC |
sgRNA名称 sgRNA name | 靶位点(拷贝数) Target location(copy number) | 核苷酸序列 Nucleotide sequence(5'-3') |
---|---|---|
sgRNA X1 | X1(72) | GCTTGGTTAGGGTGAGTGCT |
sgRNA X3 | X3(99) | TAAGTGCTGTGTGCTGCTAC |
Table 4 Sequences of sgRNA targeting mouse X chromosome multi-copy genes
sgRNA名称 sgRNA name | 靶位点(拷贝数) Target location(copy number) | 核苷酸序列 Nucleotide sequence(5'-3') |
---|---|---|
sgRNA X1 | X1(72) | GCTTGGTTAGGGTGAGTGCT |
sgRNA X3 | X3(99) | TAAGTGCTGTGTGCTGCTAC |
Fig.1 Construction and identification of CMV vector A: Schematic diagram of CMV carrier construction. B: Electrophoresis diagram of plasmid double digestion results synthesized by vector 1 and sgRNA fragments, M1 is 1000 DNA marker, M2 is 10000 DNA marker, A is the original vector 1, B is the digested vector 1, M is 10000 DNA marker, and pKMV is a plasmid synthesizing sgRNA fragment
Fig. 2 Electrophoresis diagram of verifying the efficiency of sgRNA X1 and sgRNA X3 mediated Cas9 protein cleaving target sequence in vitro a is the uncut sgRNA target sequence fragment, b is the cut sgRNA target sequence fragment, arrow indicates the cut fragment, and M is 2000 DNA marker
Fig.3 Comparison results of the number of fluorescent cells in MLTC-1 cells at different time after transfecting with CMV vector and empty vector A: Fluorescence renderings at different time points after male cells were transfected with CMV vector and empty vector. B: Proportion of green fluorescent cells at different time points after vector transfection
Fig. 4 Sequencing results of the X1 target site of the monoclonal cell mass of MLTC-1 cells after CMV vector transfection Target sites are marked in the red box, * is the matching base, and the missing space is the mutation base
转染细胞 Transfection cell | 检测筛选获得的单克隆细胞团数 Number of monoclonal cell clusters obtained by screening was detected | 突变的单克隆细胞团数 Number of mutant monoclonal cell clusters | 突变效率 Mutation efficiency/% |
---|---|---|---|
MLTC-1 | 36 | 4 | 11.1 |
Table 5 Mutation efficiency of CMV vector transfected MLTC-1 cell X1 target site
转染细胞 Transfection cell | 检测筛选获得的单克隆细胞团数 Number of monoclonal cell clusters obtained by screening was detected | 突变的单克隆细胞团数 Number of mutant monoclonal cell clusters | 突变效率 Mutation efficiency/% |
---|---|---|---|
MLTC-1 | 36 | 4 | 11.1 |
组别 Group | 注射胚胎数Number of injected embryos | 卵裂胚数Number of cleavage embryo | 卵裂率 Cleavage rate | 囊胚数Number of blastocysts | 囊胚率 Blastocyst rate | 囊胚细胞数 Number of blastocyst cells |
---|---|---|---|---|---|---|
注射 CMV 载体组 The Group of CMV vector injection | 98 | 96 | 98.0% | 57 | 58.1% a | 33.93±4.93 a |
注射空载体组 The Group of empty vector injection | 92 | 88 | 95.7% | 64 | 69.5% a | 44.66±6.18 b |
Table 6 Comparison of in vitro development efficiency of parthenogenetic embryos injected with CMV vector andempty vector
组别 Group | 注射胚胎数Number of injected embryos | 卵裂胚数Number of cleavage embryo | 卵裂率 Cleavage rate | 囊胚数Number of blastocysts | 囊胚率 Blastocyst rate | 囊胚细胞数 Number of blastocyst cells |
---|---|---|---|---|---|---|
注射 CMV 载体组 The Group of CMV vector injection | 98 | 96 | 98.0% | 57 | 58.1% a | 33.93±4.93 a |
注射空载体组 The Group of empty vector injection | 92 | 88 | 95.7% | 64 | 69.5% a | 44.66±6.18 b |
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