Identifying and retargeting transcriptional hot spots in the human genome.
Mammalian cell line development requires streamlined methodologies that will reduce both the cost and time to identify candidate cell lines. Improvements in site-specific genomic editing techniques can result in flexible, predictable, and robust cell line engineering. However, an outstanding question in the field is the specific site of integration. Here, we seek to identify productive loci within the human genome that will result in stable, high expression of heterologous DNA. Using an unbiased, random integration approach and a green fluorescent reporter construct, we identify ten single-integrant, recombinant human cell lines that exhibit stable, high-level expression. From these cell lines, eight unique corresponding integration loci were identified. These loci are concentrated in non-protein coding regions or intronic regions of protein coding genes. Expression mapping of the surrounding genes reveals minimal disruption of endogenous gene expression. Finally, we demonstrate that targeted de novo integration at one of the identified loci, the 12(th) exon-intron region of the GRIK1 gene on chromosome 21, results in superior expression and stability compared to the standard, illegitimate integration approach at levels approaching 4-fold. The information identified here along with recent advances in site-specific genomic editing techniques can lead to expedited cell line development.
Department of Chemical Engineering, The University of Texas at Austin, Austin, Texas, USA.
Joseph K
JK
Cheng
Department of Chemical Engineering, The University of Texas at Austin, Austin, Texas, USA.|Biologics Development, Bristol-Myers Squibb, Devens, MA, USA.
Amanda M
AM
Lewis
Department of Chemical Engineering, The University of Texas at Austin, Austin, Texas, USA.
Do Soon
do S
Kim
Department of Chemical Engineering, The University of Texas at Austin, Austin, Texas, USA.
Timothy
T
Dyess
Department of Chemical Engineering, The University of Texas at Austin, Austin, Texas, USA. halper@che.utexas.edu.|Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, Texas, USA. halper@che.utexas.edu.
Hal S
HS
Alper
D002460:Cell Line; D005818:Genetic Engineering / Q000379:methods*; D056426:Genetic Loci; D015894:Genome, Human; D006801:Humans; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics*; D014162:Transfection
D011994:Recombinant Proteins
CRISPR/Cas9; Cell line development; Clone selection; HT1080; Targeted integration