| 龚鱼湄 梁宏亮 韦梦影 刘向伟 杨国栋 杨新卫.基于CRISPR/Cas9系统构建精准调控Wnt信号通路的表达载体及其效果的验证[J].现代生物医学进展英文版,2016,16(31):6031-6037. |
| 基于CRISPR/Cas9系统构建精准调控Wnt信号通路的表达载体及其效果的验证 |
| Construction and Verification of the Plasmids Fine-tuning Wnt PathwayBased on CRISPR/Cas9 Gene Editing System |
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| DOI： |
| 中文关键词: CRISPR/cas9 gRNA 质粒重组 基因表达 Wnt信号通路 |
| 英文关键词: CRISPR/cas9 gRNA Recombinant plasmid Gene expression Wnt signal pathway |
| 基金项目:国家自然科学基金项目(31100979，81570232) |
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| 中文摘要: |
| 目的：构建基于CRISPR/cas9 系统调控Wnt信号通路的载体，并在细胞水平验证其调控基因表达的效率。方法：选取Wnt 信
号中负性调控分子，设计并合成能够表达靶向上述分子gRNA 的互补DNA克隆序列，BsmBI限制性内切酶酶切载体后，采用分
子克隆的方法将上述序列克隆至目的载体lenti-sgRNA-Ms2-zeo，测序正确的克隆通过Lipofectamine2000 与
lenti-Ms2-P65-HSF1-Hygro 和lenti-dcas9-VP64-blastine 共同转染入293 细胞；转染24 h后收集细胞，qRt-PCR 检测目的基因的表
达。结果：筛选了Wnt 信号通路中已知的19 个负性调控基因；针对每个基因设计了两对gRNA 序列，并构建了能够表达gRNA和
MS2 融合序列的载体，测序结果显示重组质粒的DNA 序列与预期完全相符。随机挑选了4 个表达载体与lenti-Ms2-P65-HSF1-
Hygro 和lenti-dcas9-VP64-blastine 共转进入细胞，qPCR 结果显示构建的目的载体联合lenti-Ms2-P65-HSF1-Hygro 和
lenti-dcas9-VP64-blastine 载体可以协同促进靶分子表达。结论：本研究成功构建了基于CRISPR/cas9 基因编辑系统调控Wnt信号
的载体。 |
| 英文摘要: |
| Objective:To clone the scaffold guide RNA expression vectors targeting Wnt pathway and to verify the efficacy of
these plasmids in fine-tuning Wnt pathway using an HEK293 cell model.Methods:The negative regulators of Wnt pathway were selected
in this study. Guide RNAs targeting the promoter region of these genes were designed and corresponding DNA oligos were synthesized
for cloning into lenti-sgRNA-Ms2-zeo. The plasmid construction were done by annealing the two complementary DNA sequences before
insertion into lenti-sgRNA-Ms2-zeo vectors with the enzyme BsmBI. The acquired clones are confirmed by sequencing. After confirmation,
the plasmids were co-transfected with lenti-Ms2-P65-HSF1-Hygro and lenti-dcas9-VP64-blastine into 293 cells. Expression of the
target genes were analyzed by qPCR assay.Results:Four genes were selected in this study, all of which were well-established negative
regulators of Wnt pathway. Two guide RNAs recognizing the promoter of each gene were designed and corresponding plasmids expressing
the gRNA fused with a MS2 scaffold were constructed. Sequencing results indicated that all the clones were correctly done. 4 kinds
of plasmids were randomly selected for further gene activation efficiency analysis. As expected, the gRNA expression vectors could significantly
increase the target genes when co-transfected with lenti-Ms2-P65-HSF1-Hygro and lenti-dcas9-VP64-blastine.Conclusion:The plasmids that expressed gRNAs targeting the known Wnt negative regulators were successfully constructed, which could fine-tune
the Wnt pathway by increase the target gene expression. |
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