Tuneable endogenous mammalian target complementation via multiplexed plasmid-based recombineering

Sci Rep. 2015 Nov 27;5:17432. doi: 10.1038/srep17432.

Abstract

Understanding the quantitative functional consequences of human disease mutations requires silencing of endogenous genes and expression of mutants at close to physiological levels. Changing protein levels above or below these levels is also important for system perturbation and modelling. Fast design optimization demands flexible interchangeable cassettes for endogenous gene silencing and tuneable expression. Here, we introduce 'TEMTAC', a multigene recombineering and delivery system for simultaneous siRNA-based knockdown and regulated mutant (or other variant) expression with different dynamic ranges. We show its applicability by confirming known phenotypic effects for selected mutations for BRAF, HRAS, and SHP2.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Gene Silencing*
  • Gene Transfer Techniques*
  • Genes, Reporter
  • Genetic Engineering / methods*
  • Genetic Vectors / chemistry
  • HEK293 Cells
  • HeLa Cells
  • Humans
  • Luciferases / genetics
  • Luciferases / metabolism
  • Mutation
  • Plasmids / chemistry
  • Plasmids / metabolism
  • Protein Tyrosine Phosphatase, Non-Receptor Type 11 / antagonists & inhibitors*
  • Protein Tyrosine Phosphatase, Non-Receptor Type 11 / genetics
  • Protein Tyrosine Phosphatase, Non-Receptor Type 11 / metabolism
  • Proto-Oncogene Proteins B-raf / antagonists & inhibitors*
  • Proto-Oncogene Proteins B-raf / genetics
  • Proto-Oncogene Proteins B-raf / metabolism
  • Proto-Oncogene Proteins p21(ras) / antagonists & inhibitors*
  • Proto-Oncogene Proteins p21(ras) / genetics
  • Proto-Oncogene Proteins p21(ras) / metabolism
  • RNA, Small Interfering / genetics
  • RNA, Small Interfering / metabolism

Substances

  • RNA, Small Interfering
  • Luciferases
  • BRAF protein, human
  • Proto-Oncogene Proteins B-raf
  • Protein Tyrosine Phosphatase, Non-Receptor Type 11
  • HRAS protein, human
  • Proto-Oncogene Proteins p21(ras)