Construction of DF-1 Cell Line of Stably Expressing Cas9 Protein Mediated by Lentivirus and Verification of Its Activity
【Objective】The chicken fibroblast cell line (DF-1) of stably expressing Cas9 protein was screened, and combined with the reporter vector system based on single strand annealing (SSA) repair mechanism, the Cas9 nuclease activity in the established DF-1 cell line was detected.【Method】The lentiviral ve...
Saved in:
| Main Authors: | , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Guangdong Academy of Agricultural Sciences
2023-02-01
|
| Series: | Guangdong nongye kexue |
| Subjects: | |
| Online Access: | http://gdnykx.cnjournals.org/gdnykx/ch/reader/view_abstract.aspx?file_no=202302014 |
| Tags: |
Add Tag
No Tags, Be the first to tag this record!
|
| _version_ | 1849719688659992576 |
|---|---|
| author | Xiaojiao LI Xinyu ZHU Xian ZOU Xia YAN Yanhua HE Chenglong LUO |
| author_facet | Xiaojiao LI Xinyu ZHU Xian ZOU Xia YAN Yanhua HE Chenglong LUO |
| author_sort | Xiaojiao LI |
| collection | DOAJ |
| description | 【Objective】The chicken fibroblast cell line (DF-1) of stably expressing Cas9 protein was screened, and combined with the reporter vector system based on single strand annealing (SSA) repair mechanism, the Cas9 nuclease activity in the established DF-1 cell line was detected.【Method】The lentiviral vector plasmid carrying Cas9 protein was transfected into 293T cells packaging lentivirus together with the helper plasmid. DF-1 cells were infected with lentivirus Cas9 supernatant, and DF-1 cells with stable Cas9 protein expression were obtained through antibiotic screening. The expression of Cas9 protein in positive screened DF-1 cells was verified by polymerase chain reaction (PCR) and Western blot. The sgRNA sequence of chicken ovalbumin (OVA) gene was selected and and the annealing products were cloned into pYP152 to construct sgRNA expression vector. Primers were designed at both ends of the sgRNA target site to amplify the target fragment of OVA gene. The target fragment was cloned into mCherry-SSA reporter vector pCMV-SSA-mCherry-Hind Ⅲ to destroy the expression of mCherry protein. Then the sgRNA expression vector and mCherry-SSA reporter vector were co-transfected into DF-1 cells of stably expressing Cas9 protein. Finally, the repair of mCherry expression by different stable transformants was analyzed by fluorescence microscope.【Result】27 DF-1 cell lines of stably expressing Cas9 protein were obtained after antibiotic screening, cell genome was stably transfected by PCR amplification, and the results showed that these cells contained Cas9 protein sequences. The results of Western blot assay showed that all the stable transfection cell lines expressed Cas9 protein; after co-transformation of sgRNA expression vector and mCherry-SSA reporter vector, fluorescence microscope observation showed that all the selected stable transfection cell lines could restore the expression of mCherry protein in the report vector.【Conclusion】The DF-1 cell line of stably expressing Cas9 protein with cleavage activity was successfully established, which could provide basic materials for the subsequent research on chicken functional genes on the DF-1 cell line of stably expressing Cas9 protein. |
| format | Article |
| id | doaj-art-b2b57e9492e1464e96d25ea48ba5f29e |
| institution | DOAJ |
| issn | 1004-874X |
| language | English |
| publishDate | 2023-02-01 |
| publisher | Guangdong Academy of Agricultural Sciences |
| record_format | Article |
| series | Guangdong nongye kexue |
| spelling | doaj-art-b2b57e9492e1464e96d25ea48ba5f29e2025-08-20T03:12:05ZengGuangdong Academy of Agricultural SciencesGuangdong nongye kexue1004-874X2023-02-0150212513510.16768/j.issn.1004-874X.2023.02.014202302014Construction of DF-1 Cell Line of Stably Expressing Cas9 Protein Mediated by Lentivirus and Verification of Its ActivityXiaojiao LI0Xinyu ZHU1Xian ZOU2Xia YAN3Yanhua HE4Chenglong LUO5College of Animal Science and Technology, Zhongkai University of Agriculture and Engineering, Guangzhou 510225, ChinaCollege of Animal Science and Technology, Zhongkai University of Agriculture and Engineering, Guangzhou 510225, ChinaInstitute of Animal Science, Guangdong Academy of Agricultural Sciences/State Key Laboratory of Livestock Breeding/Guangdong Key Laboratory of Livestock Breeding and Nutrition Research, Guangzhou 510640, ChinaInstitute of Animal Science, Guangdong Academy of Agricultural Sciences/State Key Laboratory of Livestock Breeding/Guangdong Key Laboratory of Livestock Breeding and Nutrition Research, Guangzhou 510640, ChinaInstitute of Animal Science, Guangdong Academy of Agricultural Sciences/State Key Laboratory of Livestock Breeding/Guangdong Key Laboratory of Livestock Breeding and Nutrition Research, Guangzhou 510640, ChinaInstitute of Animal Science, Guangdong Academy of Agricultural Sciences/State Key Laboratory of Livestock Breeding/Guangdong Key Laboratory of Livestock Breeding and Nutrition Research, Guangzhou 510640, China【Objective】The chicken fibroblast cell line (DF-1) of stably expressing Cas9 protein was screened, and combined with the reporter vector system based on single strand annealing (SSA) repair mechanism, the Cas9 nuclease activity in the established DF-1 cell line was detected.【Method】The lentiviral vector plasmid carrying Cas9 protein was transfected into 293T cells packaging lentivirus together with the helper plasmid. DF-1 cells were infected with lentivirus Cas9 supernatant, and DF-1 cells with stable Cas9 protein expression were obtained through antibiotic screening. The expression of Cas9 protein in positive screened DF-1 cells was verified by polymerase chain reaction (PCR) and Western blot. The sgRNA sequence of chicken ovalbumin (OVA) gene was selected and and the annealing products were cloned into pYP152 to construct sgRNA expression vector. Primers were designed at both ends of the sgRNA target site to amplify the target fragment of OVA gene. The target fragment was cloned into mCherry-SSA reporter vector pCMV-SSA-mCherry-Hind Ⅲ to destroy the expression of mCherry protein. Then the sgRNA expression vector and mCherry-SSA reporter vector were co-transfected into DF-1 cells of stably expressing Cas9 protein. Finally, the repair of mCherry expression by different stable transformants was analyzed by fluorescence microscope.【Result】27 DF-1 cell lines of stably expressing Cas9 protein were obtained after antibiotic screening, cell genome was stably transfected by PCR amplification, and the results showed that these cells contained Cas9 protein sequences. The results of Western blot assay showed that all the stable transfection cell lines expressed Cas9 protein; after co-transformation of sgRNA expression vector and mCherry-SSA reporter vector, fluorescence microscope observation showed that all the selected stable transfection cell lines could restore the expression of mCherry protein in the report vector.【Conclusion】The DF-1 cell line of stably expressing Cas9 protein with cleavage activity was successfully established, which could provide basic materials for the subsequent research on chicken functional genes on the DF-1 cell line of stably expressing Cas9 protein.http://gdnykx.cnjournals.org/gdnykx/ch/reader/view_abstract.aspx?file_no=202302014stable expressionlentiviruscrispr/cas9ssa repairvector constructiondf-1 |
| spellingShingle | Xiaojiao LI Xinyu ZHU Xian ZOU Xia YAN Yanhua HE Chenglong LUO Construction of DF-1 Cell Line of Stably Expressing Cas9 Protein Mediated by Lentivirus and Verification of Its Activity Guangdong nongye kexue stable expression lentivirus crispr/cas9 ssa repair vector construction df-1 |
| title | Construction of DF-1 Cell Line of Stably Expressing Cas9 Protein Mediated by Lentivirus and Verification of Its Activity |
| title_full | Construction of DF-1 Cell Line of Stably Expressing Cas9 Protein Mediated by Lentivirus and Verification of Its Activity |
| title_fullStr | Construction of DF-1 Cell Line of Stably Expressing Cas9 Protein Mediated by Lentivirus and Verification of Its Activity |
| title_full_unstemmed | Construction of DF-1 Cell Line of Stably Expressing Cas9 Protein Mediated by Lentivirus and Verification of Its Activity |
| title_short | Construction of DF-1 Cell Line of Stably Expressing Cas9 Protein Mediated by Lentivirus and Verification of Its Activity |
| title_sort | construction of df 1 cell line of stably expressing cas9 protein mediated by lentivirus and verification of its activity |
| topic | stable expression lentivirus crispr/cas9 ssa repair vector construction df-1 |
| url | http://gdnykx.cnjournals.org/gdnykx/ch/reader/view_abstract.aspx?file_no=202302014 |
| work_keys_str_mv | AT xiaojiaoli constructionofdf1celllineofstablyexpressingcas9proteinmediatedbylentivirusandverificationofitsactivity AT xinyuzhu constructionofdf1celllineofstablyexpressingcas9proteinmediatedbylentivirusandverificationofitsactivity AT xianzou constructionofdf1celllineofstablyexpressingcas9proteinmediatedbylentivirusandverificationofitsactivity AT xiayan constructionofdf1celllineofstablyexpressingcas9proteinmediatedbylentivirusandverificationofitsactivity AT yanhuahe constructionofdf1celllineofstablyexpressingcas9proteinmediatedbylentivirusandverificationofitsactivity AT chenglongluo constructionofdf1celllineofstablyexpressingcas9proteinmediatedbylentivirusandverificationofitsactivity |