Validation of reliable safe harbor locus for efficient porcine transgenesis.

Funct Integr Genomics

Key Laboratory of Applied Technology On Green-Eco-Healthy Animal Husbandry of Zhejiang Province, China-Australia Joint Laboratory for Animal Health Big Data Analytics, Zhejiang Provincial Engineering Laboratory for Animal Health Inspection and Internet Technology, College of Animal Science and Technology, College of Veterinary Medicine, Zhejiang A&F University, 666 Wusu street, Lin'an District, Hangzhou, 311300, Zhejiang, China.

Published: August 2022

AI Article Synopsis

  • Transgenic technology is commonly used in biomedical and agricultural sectors, but traditional methods can lead to random gene integration with uncertain outcomes.
  • This study focused on identifying safe harbor (SH) loci for efficient gene integration in pigs, using CRISPR/Cas9 to test four different SH loci.
  • Results indicated that the COL1A1 and ROSA26 loci provided higher expression of a reporter gene (EGFP) and were found to be safer, with minimal impact on neighboring genes and no off-target effects.

Article Abstract

Transgenic technology is now widely used in biomedical and agricultural fields. Transgenesis is commonly achieved through random integration which might cause some uncertain consequences. The site-specific integration could avoid this disadvantage. This study aimed to screen and validate the best safe harbor (SH) locus for efficient porcine transgenesis. First, the cells carrying the EGFP reporter construct at four different SH loci (ROSA26, AAVS1, H11 and COL1A1) were achieved through CRSIPR/Cas9-mediated HDR. At the COL1A1 and ROSA26 loci, a higher mRNA and protein expression of EGFP was detected, and it was correlated with a lower level of DNA methylation of the EGFP promoter, hEF1α. A decreased H3K27me3 modification of the hEF1α promoter at the COL1A1 locus was also detected. For the safety of transgenesis at different SH locus, we found that transgenesis could relatively alter the expression of the adjacent endogenous genes, but the influence was limited. We also did not observe any off-target cleavage for the selected sgRNAs of the COL1A1 and ROSA26 loci. In conclusion, the COL1A1 and ROSA26 were confirmed to be the best two SH loci with the COL1A1 being more competitive for porcine transgenesis. This work would greatly facilitate porcine genome engineering and transgenic pig production.

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Source
http://dx.doi.org/10.1007/s10142-022-00859-3DOI Listing

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