Format

Send to

Choose Destination
Nucleic Acids Res. 2017 Jun 20;45(11):e102. doi: 10.1093/nar/gkx183.

A platform for functional assessment of large variant libraries in mammalian cells.

Author information

1
Department of Genome Sciences, University of Washington, Seattle, WA 98195, USA.
2
Department of Bioengineering, University of Washington, Seattle, WA 98195, USA.

Abstract

Sequencing-based, massively parallel genetic assays have revolutionized our ability to quantify the relationship between many genotypes and a phenotype of interest. Unfortunately, variant library expression platforms in mammalian cells are far from ideal, hindering the study of human gene variants in their physiologically relevant cellular contexts. Here, we describe a platform for phenotyping variant libraries in transfectable mammalian cell lines in two steps. First, a landing pad cell line with a genomically integrated, Tet-inducible cassette containing a Bxb1 recombination site is created. Second, a single variant from a library of transfected, promoter-less plasmids is recombined into the landing pad in each cell. Thus, every cell in the recombined pool expresses a single variant, allowing for parallel, sequencing-based assessment of variant effect. We describe a method for incorporating a single landing pad into a defined site of a cell line of interest, and show that our approach can be used generate more than 20 000 recombinant cells in a single experiment. Finally, we use our platform in combination with a sequencing-based assay to explore the N-end rule by simultaneously measuring the effects of all possible N-terminal amino acids on protein expression.

PMID:
28335006
PMCID:
PMC5499817
DOI:
10.1093/nar/gkx183
[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for Silverchair Information Systems Icon for PubMed Central
Loading ...
Support Center