In vivo expression of signaling proteins in reconstituted NK cells

J Immunol Methods. 2009 Jan 30;340(2):158-63. doi: 10.1016/j.jim.2008.10.014. Epub 2008 Nov 24.

Abstract

Natural Killer cells are cells of the innate immune system that are important for the recognition and clearance of virally infected cells or tumors. Examination of the development and signaling of these cells has been severely hampered due to an inability to over-express proteins in these cells. We developed a novel technique to generate NK cells in vivo, all of which express a gene of interest. IL2Rgamma(c)(-/-)/Rag2(-/-) mice do not develop NK cells due to the lack of IL15 signaling. We infected bone marrow from IL2Rgamma(c)(-/-)/Rag2(-/-) mice with a retroviral construct encoding EGFP and IL2Rgamma(c) connected by an IRES. NK cells selectively developed through expression of IL2Rgamma(c) and 100% of these NK cells were found to be EGFP(+). In order to test the utilization of this method to examine the function of biologically relevant proteins, constitutively active PI3K p110gamma and p110delta isoforms were over-expressed in this system. Constitutively active p110gamma revealed profound effects on NK cell development and function in vivo while p110delta had little effect.

MeSH terms

  • Animals
  • Gene Expression / genetics*
  • Genes, Reporter / genetics*
  • Killer Cells, Natural / metabolism*
  • Mice
  • Mice, Inbred C57BL
  • Proteins / analysis*
  • Proteins / genetics
  • Proteins / metabolism*

Substances

  • Proteins