A platform for functional assessment of large variant libraries in mammalian cells.
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.
Department of Genome Sciences, University of Washington, Seattle, WA 98195, USA.
Kenneth A
KA
Matreyek
Department of Bioengineering, University of Washington, Seattle, WA 98195, USA.
Jason J
JJ
Stephany
Department of Genome Sciences, University of Washington, Seattle, WA 98195, USA.|Department of Bioengineering, University of Washington, Seattle, WA 98195, USA.
Douglas M
DM
Fowler
D001483:Base Sequence; D000229:Dependovirus / Q000235:genetics; D015870:Gene Expression; D015723:Gene Library; D056726:Genetic Association Studies; D014644:Genetic Variation; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D057809:HEK293 Cells; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D010641:Phenotype; D055550:Protein Stability; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D014162:Transfection
D011994:Recombinant Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins