The collagen type I alpha 1 chain gene is an alternative safe harbor locus in the porcine genome
Efficient and stable expression of foreign genes in cells and transgenic animals is important for gain-of-function studies and the establishment of bioreactors. Safe harbor loci in the animal genome enable consistent overexpression of foreign genes, without side effects. However, relatively few safe...
| Main Authors: | , , , , , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Elsevier
2023-01-01
|
| Series: | Journal of Integrative Agriculture |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2095311922001836 |
| _version_ | 1797960976435249152 |
|---|---|
| author | Guang-ming XIANG Xiu-ling ZHANG Chang-jiang XU Zi-yao FAN Kui XU Nan WANG Yue WANG Jing-jing CHE Song-song XU Yu-lian MU Kui LI Zhi-guo LIU |
| author_facet | Guang-ming XIANG Xiu-ling ZHANG Chang-jiang XU Zi-yao FAN Kui XU Nan WANG Yue WANG Jing-jing CHE Song-song XU Yu-lian MU Kui LI Zhi-guo LIU |
| author_sort | Guang-ming XIANG |
| collection | DOAJ |
| description | Efficient and stable expression of foreign genes in cells and transgenic animals is important for gain-of-function studies and the establishment of bioreactors. Safe harbor loci in the animal genome enable consistent overexpression of foreign genes, without side effects. However, relatively few safe harbor loci are available in pigs, a fact which has impeded the development of multi-transgenic pig research. We report a strategy for efficient transgene knock-in in the endogenous collagen type I alpha 1 chain (COL1A1) gene using the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system. After the knock-in of a 2A peptide-green fluorescence protein (2A-GFP) transgene in the last codon of COL1A1 in multiple porcine cells, including porcine kidney epithelial (PK15), porcine embryonic fibroblast (PEF) and porcine intestinal epithelial (IPI-2I) cells, quantitative PCR (qPCR), Western blotting, RNA-seq and CCK8 assay were performed to assess the safety of COL1A1 locus. The qPCR results showed that the GFP knock-in had no effect (P=0.29, P=0.66 and P=0.20 for PK15, PEF and IPI-2I cells, respectively) on the mRNA expression of COL1A1 gene. Similarly, no significant differences (P=0.64, P=0.48 and P=0.80 for PK15, PEF and IPI-2I cells, respectively) were found between the GFP knock-in and wild type cells by Western blotting. RNA-seq results revealed that the transcriptome of GFP knock-in PEF cells had a significant positive correlation (P<2.2e–16) with that of the wild type cells, indicating that the GFP knock-in did not alter the global expression of endogenous genes. Furthermore, the CCK8 assay showed that the GFP knock-in events had no adverse effects (P24h=0.31, P48h=0.96, P72h=0.24, P96h=0.17, and P120h=0.38) on cell proliferation of PK15 cells. These results indicate that the COL1A1 locus can be used as a safe harbor for foreign genes knock-in into the pig genome and can be broadly applied to farm animal breeding and biomedical model establishment. |
| first_indexed | 2024-04-11T00:53:19Z |
| format | Article |
| id | doaj.art-730aff45c1ec4bbf9587039fddc1b488 |
| institution | Directory Open Access Journal |
| issn | 2095-3119 |
| language | English |
| last_indexed | 2024-04-11T00:53:19Z |
| publishDate | 2023-01-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Journal of Integrative Agriculture |
| spelling | doaj.art-730aff45c1ec4bbf9587039fddc1b4882023-01-05T08:36:32ZengElsevierJournal of Integrative Agriculture2095-31192023-01-01221202213The collagen type I alpha 1 chain gene is an alternative safe harbor locus in the porcine genomeGuang-ming XIANG0Xiu-ling ZHANG1Chang-jiang XU2Zi-yao FAN3Kui XU4Nan WANG5Yue WANG6Jing-jing CHE7Song-song XU8Yu-lian MU9Kui LI10Zhi-guo LIU11State Key Laboratory of Animal Nutrition/Key Laboratory of Animal Genetics Breeding and Reproduction of Ministry of Agriculture and Rural Affairs of China, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, P.R.ChinaState Key Laboratory of Animal Nutrition/Key Laboratory of Animal Genetics Breeding and Reproduction of Ministry of Agriculture and Rural Affairs of China, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, P.R.ChinaState Key Laboratory of Animal Nutrition/Key Laboratory of Animal Genetics Breeding and Reproduction of Ministry of Agriculture and Rural Affairs of China, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, P.R.ChinaState Key Laboratory of Animal Nutrition/Key Laboratory of Animal Genetics Breeding and Reproduction of Ministry of Agriculture and Rural Affairs of China, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, P.R.ChinaState Key Laboratory of Animal Nutrition/Key Laboratory of Animal Genetics Breeding and Reproduction of Ministry of Agriculture and Rural Affairs of China, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, P.R.ChinaState Key Laboratory of Animal Nutrition/Key Laboratory of Animal Genetics Breeding and Reproduction of Ministry of Agriculture and Rural Affairs of China, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, P.R.ChinaState Key Laboratory of Animal Nutrition/Key Laboratory of Animal Genetics Breeding and Reproduction of Ministry of Agriculture and Rural Affairs of China, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, P.R.China; School of Life Science and Engineering, Foshan University, Foshan 528231, P.R.ChinaState Key Laboratory of Animal Nutrition/Key Laboratory of Animal Genetics Breeding and Reproduction of Ministry of Agriculture and Rural Affairs of China, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, P.R.ChinaState Key Laboratory of Animal Nutrition/Key Laboratory of Animal Genetics Breeding and Reproduction of Ministry of Agriculture and Rural Affairs of China, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, P.R.China; Genome Analysis Laboratory of the Ministry of Agriculture and Rural Affairs, Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences, Shenzhen 518120, P.R.ChinaState Key Laboratory of Animal Nutrition/Key Laboratory of Animal Genetics Breeding and Reproduction of Ministry of Agriculture and Rural Affairs of China, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, P.R.ChinaState Key Laboratory of Animal Nutrition/Key Laboratory of Animal Genetics Breeding and Reproduction of Ministry of Agriculture and Rural Affairs of China, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, P.R.China; Genome Analysis Laboratory of the Ministry of Agriculture and Rural Affairs, Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences, Shenzhen 518120, P.R.ChinaState Key Laboratory of Animal Nutrition/Key Laboratory of Animal Genetics Breeding and Reproduction of Ministry of Agriculture and Rural Affairs of China, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, P.R.China; Correspondence LIU Zhi-guoEfficient and stable expression of foreign genes in cells and transgenic animals is important for gain-of-function studies and the establishment of bioreactors. Safe harbor loci in the animal genome enable consistent overexpression of foreign genes, without side effects. However, relatively few safe harbor loci are available in pigs, a fact which has impeded the development of multi-transgenic pig research. We report a strategy for efficient transgene knock-in in the endogenous collagen type I alpha 1 chain (COL1A1) gene using the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system. After the knock-in of a 2A peptide-green fluorescence protein (2A-GFP) transgene in the last codon of COL1A1 in multiple porcine cells, including porcine kidney epithelial (PK15), porcine embryonic fibroblast (PEF) and porcine intestinal epithelial (IPI-2I) cells, quantitative PCR (qPCR), Western blotting, RNA-seq and CCK8 assay were performed to assess the safety of COL1A1 locus. The qPCR results showed that the GFP knock-in had no effect (P=0.29, P=0.66 and P=0.20 for PK15, PEF and IPI-2I cells, respectively) on the mRNA expression of COL1A1 gene. Similarly, no significant differences (P=0.64, P=0.48 and P=0.80 for PK15, PEF and IPI-2I cells, respectively) were found between the GFP knock-in and wild type cells by Western blotting. RNA-seq results revealed that the transcriptome of GFP knock-in PEF cells had a significant positive correlation (P<2.2e–16) with that of the wild type cells, indicating that the GFP knock-in did not alter the global expression of endogenous genes. Furthermore, the CCK8 assay showed that the GFP knock-in events had no adverse effects (P24h=0.31, P48h=0.96, P72h=0.24, P96h=0.17, and P120h=0.38) on cell proliferation of PK15 cells. These results indicate that the COL1A1 locus can be used as a safe harbor for foreign genes knock-in into the pig genome and can be broadly applied to farm animal breeding and biomedical model establishment.http://www.sciencedirect.com/science/article/pii/S2095311922001836COL1A1 genesafe harborknock-inCRISPR/Cas9pig |
| spellingShingle | Guang-ming XIANG Xiu-ling ZHANG Chang-jiang XU Zi-yao FAN Kui XU Nan WANG Yue WANG Jing-jing CHE Song-song XU Yu-lian MU Kui LI Zhi-guo LIU The collagen type I alpha 1 chain gene is an alternative safe harbor locus in the porcine genome Journal of Integrative Agriculture COL1A1 gene safe harbor knock-in CRISPR/Cas9 pig |
| title | The collagen type I alpha 1 chain gene is an alternative safe harbor locus in the porcine genome |
| title_full | The collagen type I alpha 1 chain gene is an alternative safe harbor locus in the porcine genome |
| title_fullStr | The collagen type I alpha 1 chain gene is an alternative safe harbor locus in the porcine genome |
| title_full_unstemmed | The collagen type I alpha 1 chain gene is an alternative safe harbor locus in the porcine genome |
| title_short | The collagen type I alpha 1 chain gene is an alternative safe harbor locus in the porcine genome |
| title_sort | collagen type i alpha 1 chain gene is an alternative safe harbor locus in the porcine genome |
| topic | COL1A1 gene safe harbor knock-in CRISPR/Cas9 pig |
| url | http://www.sciencedirect.com/science/article/pii/S2095311922001836 |
| work_keys_str_mv | AT guangmingxiang thecollagentypeialpha1chaingeneisanalternativesafeharborlocusintheporcinegenome AT xiulingzhang thecollagentypeialpha1chaingeneisanalternativesafeharborlocusintheporcinegenome AT changjiangxu thecollagentypeialpha1chaingeneisanalternativesafeharborlocusintheporcinegenome AT ziyaofan thecollagentypeialpha1chaingeneisanalternativesafeharborlocusintheporcinegenome AT kuixu thecollagentypeialpha1chaingeneisanalternativesafeharborlocusintheporcinegenome AT nanwang thecollagentypeialpha1chaingeneisanalternativesafeharborlocusintheporcinegenome AT yuewang thecollagentypeialpha1chaingeneisanalternativesafeharborlocusintheporcinegenome AT jingjingche thecollagentypeialpha1chaingeneisanalternativesafeharborlocusintheporcinegenome AT songsongxu thecollagentypeialpha1chaingeneisanalternativesafeharborlocusintheporcinegenome AT yulianmu thecollagentypeialpha1chaingeneisanalternativesafeharborlocusintheporcinegenome AT kuili thecollagentypeialpha1chaingeneisanalternativesafeharborlocusintheporcinegenome AT zhiguoliu thecollagentypeialpha1chaingeneisanalternativesafeharborlocusintheporcinegenome AT guangmingxiang collagentypeialpha1chaingeneisanalternativesafeharborlocusintheporcinegenome AT xiulingzhang collagentypeialpha1chaingeneisanalternativesafeharborlocusintheporcinegenome AT changjiangxu collagentypeialpha1chaingeneisanalternativesafeharborlocusintheporcinegenome AT ziyaofan collagentypeialpha1chaingeneisanalternativesafeharborlocusintheporcinegenome AT kuixu collagentypeialpha1chaingeneisanalternativesafeharborlocusintheporcinegenome AT nanwang collagentypeialpha1chaingeneisanalternativesafeharborlocusintheporcinegenome AT yuewang collagentypeialpha1chaingeneisanalternativesafeharborlocusintheporcinegenome AT jingjingche collagentypeialpha1chaingeneisanalternativesafeharborlocusintheporcinegenome AT songsongxu collagentypeialpha1chaingeneisanalternativesafeharborlocusintheporcinegenome AT yulianmu collagentypeialpha1chaingeneisanalternativesafeharborlocusintheporcinegenome AT kuili collagentypeialpha1chaingeneisanalternativesafeharborlocusintheporcinegenome AT zhiguoliu collagentypeialpha1chaingeneisanalternativesafeharborlocusintheporcinegenome |