|
KMID : 1094720210260050795
|
|
Biotechnology and Bioprocess Engineering
2021 Volume.26 No. 5 p.795 ~ p.803
|
|
|
Cre/Lox-based RMCE for Site-specific Integration in CHO Cells
|
|
Kim Jae-Won
Lee Yun-Haeng
Kuk Myeong-Uk
Hwang Su-Young
Kwon Hyung-Wook
Park Joon-Tae
|
|
|
Abstract
|
|
|
|
Traditional cell line development is based on random genomic integration of transgenes. Random integration leads to unpredictable expression and results in clonal heterogeneity requiring a tedious screening procedure. Therefore, a new strategy is needed to establish clones that exhibit stable transgene expression. Here, we performed CRISPR/Cas9-mediated site-specific integration (SSI) to incorporate a landing pad (LP; containing mCherry) at a genomic hotspot (Fer1L4) allowing stable and strong expression. Site-specific integration of LP on Fer1L4 was demonstrated by sequencing results representing the swapped sequences in mCherry-expressing cells. We then performed Cre/Lox-based recombinase-mediated cassette exchange (RMCE) to exchange LP with a targeting vector (TV; containing GFP) in clones established by CRISPR/Cas9-mediated SSI. The success of Cre/Lox-based RMCE was evidenced by sequencing results representing the swapped sequences in GFP-expressing cells. Furthermore, the swapped clones expressing GFP was enriched up to 80%, indicating that the efficiency of Cre/Lox-based RMCE would be sufficient to swap pre-existing cassettes with gene-of-interest (GOI). Taken together, our study provides a new platform for Cre/Lox-based RMCE to iteratively establish stable clones from existing ones previously established by SSI at a genomic hotspot.
|
|
|
KEYWORD
|
|
|
Cre/Lox-based RMCE, CRISPR/Cas9, site-specific integration, cell line development
|
|
|
FullTexts / Linksout information
|
|
|
|
|
|
Listed journal information
|
|
 
|