A speedy method to detect inserted DNA fragment in cell lines transfected with retroviral vectors

Liang H. Ding, Emi Iimura, Kaoru Saijo, Hirofumi Hamada, Tadao Ohno

Research output: Contribution to journalArticle

1 Scopus citations

Abstract

Cells transfected by retroviral vectors are brought in a gene of particular interest and are very useful in a variety of experiments. It is essential to testify that the DNA fragment was successfully introduced into the cells together with the retroviral vectors. Polymerase chain reaction is believed to be a fast and convenient method for this purpose when using primers flanking the cloning site of the inserted DNA. Unfortunately, a single PCR reaction often fails to amplify the targeted fragment because of the existence of endogenous virus DNA in cell genome. However, in this study we conducted a procedure for a single PCR, using vector-specific primers as well as a nested PCR, and successfully detected the DNA fragments cloned in MFG retroviral vectors in 22 transfected cell lines. We also proved that real time quantitative PCR in combination with MFG-specific primer is useful to determine copy number of the retroviral vector in murine producer cell lines.

Original languageEnglish (US)
Pages (from-to)243-252
Number of pages10
JournalCytotechnology
Volume34
Issue number3
DOIs
StatePublished - Dec 1 2000

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Keywords

  • PCR
  • Quantitative PCR
  • Retroviral vector
  • Retroviral vector specific primer

ASJC Scopus subject areas

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

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