Determining the effects of loss of function mutations in human cell lines

STAR Protoc. 2022 Mar 15;3(2):101232. doi: 10.1016/j.xpro.2022.101232. eCollection 2022 Jun 17.

Abstract

Quantifying differences in the amount of protein and mRNA caused by missense mutations in a gene of interest can be challenging, especially when using patient-derived primary cells, which are intrinsically variable. In this protocol, we describe how to culture patient-derived lymphoblast and fibroblast cell lines for later mRNA and protein quantification. We also describe the steps to examine variants of PUM1 in HEK293T cells, but the protocol can be applied to other proteins of interest. For complete details on the use and execution of this protocol, please refer to Gennarino et al. (2018).

Keywords: Cell culture; Cell isolation; Genetics; Model Organisms; Protein expression and purification.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • HEK293 Cells
  • Humans
  • Loss of Function Mutation*
  • Mutation
  • Proteins*
  • RNA, Messenger / genetics
  • RNA-Binding Proteins

Substances

  • PUM1 protein, human
  • Proteins
  • RNA, Messenger
  • RNA-Binding Proteins