Knock-in fibroblasts and transgenic blastocysts for expression of human FGF2 in the bovine β-casein gene locus using CRISPR/Cas9 nuclease-mediated homologous recombination
Autor: | Hong-Gu Lee, Se Eun Kim, Se Pill Park, Min Jee Park, Man-Jong Kang, Jiwoo Kim, Eun-Young Kim, Yeong Ji Kim, Young-Hee Jeong |
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Rok vydání: | 2015 |
Předmět: |
0301 basic medicine
Transgene Blotting Western Gene Expression Biology Cell Line Animals Genetically Modified Mice 03 medical and health sciences Exon Gene knockin Gene expression Animals Humans CRISPR Amino Acid Sequence Homologous Recombination Enhancer Gene Cells Cultured Regulation of gene expression Base Sequence Reverse Transcriptase Polymerase Chain Reaction Caseins Cell Biology Fibroblasts Endonucleases Molecular biology Blastocyst 030104 developmental biology Cattle Fibroblast Growth Factor 2 CRISPR-Cas Systems Genetic Engineering Developmental Biology |
Zdroj: | Zygote. 24:442-456 |
ISSN: | 1469-8730 0967-1994 |
Popis: | SummaryMany transgenic domestic animals have been developed to produce therapeutic proteins in the mammary gland, and this approach is one of the most important methods for agricultural and biomedical applications. However, expression and secretion of a protein varies because transgenes are integrated at random sites in the genome. In addition, distal enhancers are very important for transcriptional gene regulation and tissue-specific gene expression. Development of a vector system regulated accurately in the genome is needed to improve production of therapeutic proteins. The objective of this study was to develop a knock-in system for expression of human fibroblast growth factor 2 (FGF2) in the bovine β-casein gene locus. The F2A sequence was fused to the human FGF2 gene and inserted into exon 3 of the β-casein gene. We detected expression of human FGF2 mRNA in the HC11 mouse mammary epithelial cells by RT-PCR and human FGF2 protein in the culture media using western blot analysis when the knock-in vector was introduced. We transfected the knock-in vector into bovine ear fibroblasts and produced knock-in fibroblasts using the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system. Moreover, the CRISPR/Cas9 system was more efficient than conventional methods. In addition, we produced knock-in blastocysts by somatic cell nuclear transfer using the knock-in fibroblasts. Our knock-in fibroblasts may help to create cloned embryos for development of transgenic dairy cattle expressing human FGF2 protein in the mammary gland via the expression system of the bovine β-casein gene. |
Databáze: | OpenAIRE |
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