Abstract
The genetic modification of cattle has many agricultural and biomedical applications. However, random integration often leads to the unstable or differentially expression of the exogenous genes, which limit the application and development of transgenic technologies. Finding a safe locus suitable for site-specific insertion and efficient expression of exogenous genes is a good way to overcome these hurdles. In this study, we efficiently integrated three targeted vector into the cattle Rosa26 (cRosa26) by CRISPR/Cas9 technology in which EGFP was driven by CAG, EF1a, PGK and cRosa26 endogenous promoter respectively. The CRISPR/Cas9 knock-in system allows highly efficient gene insertion of different expression units at the cRosa26 locus. We also find that in the four cell lines, EGFP was stable expressed at different times, and the CAG promoter has the highest activity to activate the expression of EGFP, when compared with the cRosa26, EF1a and PGK promoter. Our results proved that cRosa26 was a locus that could integrate different expression units efficiently, and supported the friendly expression of different expression units. Our findings described here will be useful for a variety of studies using cattle.
Funder
National Natural Science Foundation of China
Agricultural Variety Improvement Project of Shandong Province
Jilin Province Education Department Science and Technology Research Project
Publisher
Public Library of Science (PLoS)
Reference31 articles.
1. Cloned transgenic cattle produce milk with higher levels of beta-casein and kappa-casein;B Brophy;Nat Biotechnol,2003
2. Large scale production of recombinant human lactoferrin in the milk of transgenic cows;PV Berkel;Nat Biotechnol,2002
3. Large-scale production of recombinant human lactoferrin from high-expression, marker-free transgenic cloned cows;M Wang;Rep,2017
4. Genetically enhanced cows resist intramammary Staphylococcus aureus infection;RJ Wall;Nat Biotechnol,2005
5. Tale nickase-mediated sp110 knockin endows cattle with increased resistance to tuberculosis;H Wu;P Natl Acad Sci USA,2015