Protein function analysis

Rapid, cell-based SiRNA-mediated ablation of endogenous expression with simultaneous ectopic replacement

Brett C. DiNatale, Gary H. Perdew

Research output: Contribution to journalArticle

4 Citations (Scopus)

Abstract

Current methods for determining and dissecting the function of a specific protein within a cell are laborious and limiting. We have developed a method by which endogenous protein levels are rapidly ablated and simultaneous expression of a designed, inserted variant takes place in the native setting. Through optimized electroporation, siRNA oligonucleotides and codon-optimized coding sequence containing vectors can be co-transfected, leading to expression of ectopic mRNA not targeted by siRNA. Using the commonly encountered MCF-7 breast cancer cell line, we were able to reach 90% transfection efficiency. Under these conditions, siRNA oligonucleotides were transfected simultaneously with a codon-optimized, cDNA containing vector encoding the AHR protein. Thus, endogenous protein was ablated while the designed protein was fully expressed in the native environment. The codonoptimized AHR was shown to be fully functional in its ability to induce CYP1A1 transcription and to rescue a B[a]P-susceptible phenotype.

Original languageEnglish (US)
Pages (from-to)95-100
Number of pages6
JournalCytotechnology
Volume62
Issue number2
DOIs
StatePublished - Apr 1 2010

Fingerprint

Ablation
Proteins
Small Interfering RNA
Oligonucleotides
Codon
Cytochrome P-450 CYP1A1
Electroporation
Transcription
Transfection
Complementary DNA
Cells
Breast Neoplasms
Phenotype
Cell Line
Messenger RNA

All Science Journal Classification (ASJC) codes

  • Biotechnology
  • Cell Biology
  • Clinical Biochemistry
  • Bioengineering
  • Biomedical Engineering

Cite this

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Protein function analysis : Rapid, cell-based SiRNA-mediated ablation of endogenous expression with simultaneous ectopic replacement. / DiNatale, Brett C.; Perdew, Gary H.

In: Cytotechnology, Vol. 62, No. 2, 01.04.2010, p. 95-100.

Research output: Contribution to journalArticle

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