Nima Dehdilani, Lena Goshayeshi, Sara Yousefi Taemeh, Ahmad Reza Bahrami, Sylvie Rival Gervier, Bertrand Pain, Hesam Dehghani
{"title":"整合组学和CRISPR技术鉴定和验证鸡基因组安全港位点。","authors":"Nima Dehdilani, Lena Goshayeshi, Sara Yousefi Taemeh, Ahmad Reza Bahrami, Sylvie Rival Gervier, Bertrand Pain, Hesam Dehghani","doi":"10.1186/s12575-023-00210-5","DOIUrl":null,"url":null,"abstract":"<p><strong>Background: </strong>One of the most prominent questions in the field of transgenesis is 'Where in the genome to integrate a transgene?'. Escape from epigenetic silencing and promoter shutdown of the transgene needs reliable genomic safe harbor (GSH) loci. Advances in genome engineering technologies combined with multi-omics bioinformatics data have enabled rational evaluation of GSH loci in the host genome. Currently, no validated GSH loci have been evaluated in the chicken genome.</p><p><strong>Results: </strong>Here, we analyzed and experimentally examined two GSH loci in the genome of chicken cells. To this end, putative GSH loci including chicken HIPP-like (cHIPP; between DRG1 and EIF4ENIF1 genes) and chicken ROSA-like (cROSA; upstream of the THUMPD3 gene) were predicted using multi-omics bioinformatics data. Then, the durable expression of the transgene was validated by experimental characterization of continuously-cultured isogenous cell clones harboring DsRed2-ΔCMV-EGFP cassette in the predicted loci. The weakened form of the CMV promoter (ΔCMV) allowed the precise evaluation of GSH loci in a locus-dependent manner compared to the full-length CMV promoter.</p><p><strong>Conclusions: </strong>cHIPP and cROSA loci introduced in this study can be reliably exploited for consistent bio-manufacturing of recombinant proteins in the genetically-engineered chickens. Also, results showed that the genomic context dictates the expression of transgene controlled by ΔCMV in GSH loci.</p>","PeriodicalId":8960,"journal":{"name":"Biological Procedures Online","volume":"25 1","pages":"18"},"PeriodicalIF":3.7000,"publicationDate":"2023-06-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10290409/pdf/","citationCount":"1","resultStr":"{\"title\":\"Integrating Omics and CRISPR Technology for Identification and Verification of Genomic Safe Harbor Loci in the Chicken Genome.\",\"authors\":\"Nima Dehdilani, Lena Goshayeshi, Sara Yousefi Taemeh, Ahmad Reza Bahrami, Sylvie Rival Gervier, Bertrand Pain, Hesam Dehghani\",\"doi\":\"10.1186/s12575-023-00210-5\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><strong>Background: </strong>One of the most prominent questions in the field of transgenesis is 'Where in the genome to integrate a transgene?'. Escape from epigenetic silencing and promoter shutdown of the transgene needs reliable genomic safe harbor (GSH) loci. Advances in genome engineering technologies combined with multi-omics bioinformatics data have enabled rational evaluation of GSH loci in the host genome. Currently, no validated GSH loci have been evaluated in the chicken genome.</p><p><strong>Results: </strong>Here, we analyzed and experimentally examined two GSH loci in the genome of chicken cells. To this end, putative GSH loci including chicken HIPP-like (cHIPP; between DRG1 and EIF4ENIF1 genes) and chicken ROSA-like (cROSA; upstream of the THUMPD3 gene) were predicted using multi-omics bioinformatics data. Then, the durable expression of the transgene was validated by experimental characterization of continuously-cultured isogenous cell clones harboring DsRed2-ΔCMV-EGFP cassette in the predicted loci. The weakened form of the CMV promoter (ΔCMV) allowed the precise evaluation of GSH loci in a locus-dependent manner compared to the full-length CMV promoter.</p><p><strong>Conclusions: </strong>cHIPP and cROSA loci introduced in this study can be reliably exploited for consistent bio-manufacturing of recombinant proteins in the genetically-engineered chickens. Also, results showed that the genomic context dictates the expression of transgene controlled by ΔCMV in GSH loci.</p>\",\"PeriodicalId\":8960,\"journal\":{\"name\":\"Biological Procedures Online\",\"volume\":\"25 1\",\"pages\":\"18\"},\"PeriodicalIF\":3.7000,\"publicationDate\":\"2023-06-24\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10290409/pdf/\",\"citationCount\":\"1\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Biological Procedures Online\",\"FirstCategoryId\":\"99\",\"ListUrlMain\":\"https://doi.org/10.1186/s12575-023-00210-5\",\"RegionNum\":3,\"RegionCategory\":\"生物学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q1\",\"JCRName\":\"BIOCHEMICAL RESEARCH METHODS\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Biological Procedures Online","FirstCategoryId":"99","ListUrlMain":"https://doi.org/10.1186/s12575-023-00210-5","RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"BIOCHEMICAL RESEARCH METHODS","Score":null,"Total":0}
Integrating Omics and CRISPR Technology for Identification and Verification of Genomic Safe Harbor Loci in the Chicken Genome.
Background: One of the most prominent questions in the field of transgenesis is 'Where in the genome to integrate a transgene?'. Escape from epigenetic silencing and promoter shutdown of the transgene needs reliable genomic safe harbor (GSH) loci. Advances in genome engineering technologies combined with multi-omics bioinformatics data have enabled rational evaluation of GSH loci in the host genome. Currently, no validated GSH loci have been evaluated in the chicken genome.
Results: Here, we analyzed and experimentally examined two GSH loci in the genome of chicken cells. To this end, putative GSH loci including chicken HIPP-like (cHIPP; between DRG1 and EIF4ENIF1 genes) and chicken ROSA-like (cROSA; upstream of the THUMPD3 gene) were predicted using multi-omics bioinformatics data. Then, the durable expression of the transgene was validated by experimental characterization of continuously-cultured isogenous cell clones harboring DsRed2-ΔCMV-EGFP cassette in the predicted loci. The weakened form of the CMV promoter (ΔCMV) allowed the precise evaluation of GSH loci in a locus-dependent manner compared to the full-length CMV promoter.
Conclusions: cHIPP and cROSA loci introduced in this study can be reliably exploited for consistent bio-manufacturing of recombinant proteins in the genetically-engineered chickens. Also, results showed that the genomic context dictates the expression of transgene controlled by ΔCMV in GSH loci.
期刊介绍：
iological Procedures Online publishes articles that improve access to techniques and methods in the medical and biological sciences.
We are also interested in short but important research discoveries, such as new animal disease models.
Topics of interest include, but are not limited to:
Reports of new research techniques and applications of existing techniques
Technical analyses of research techniques and published reports
Validity analyses of research methods and approaches to judging the validity of research reports
Application of common research methods
Reviews of existing techniques
Novel/important product information
Biological Procedures Online places emphasis on multidisciplinary approaches that integrate methodologies from medicine, biology, chemistry, imaging, engineering, bioinformatics, computer science, and systems analysis.
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