[Feasibility of real-time quantitative PCR in assessing the efficiency of gene transfection in vivo].
To explore feasibility of real-time quantitative PCR in assessing efficiency of gene transfection, clonal PCR was employed to analyze efficiency of retroviral-mediated neo gene transfection in primary myoblast, simultaneous real-time PCR were performed for estimation of transfection efficiency; for measuring integrated gene copy number per cell, linear amplification mediated-PCR (LAM-PCR) and retroviral 5'LTR integration analysis also were used. The results showed that: (1) the data from clonal PCR are similar as that from real-time PCR in low efficiency of transfection (< 36%); but in high efficiency of transfection, it is significantly differentiation between clonal PCR and real-time PCR. (2) One copy of transduced gene per cell was observed in retroviral-mediated gene transfection in primary myoblast. It is concluded that real-time PCR can be used to estimate gene transfer vector in vivo, but it is not available for assessing gene transfection in vitro, because high efficiency of transfection could be obtained in most of gene transfection in vitro.
Gao
Chun-Ji
CJ
Department of Hematology, General Hospital of PLA, Beijing 100853, China. gaochunji@hotmail.com
D000818:Animals; D005822:Genetic Vectors / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D012190:Retroviridae / Q000235:genetics; D017422:Sequence Analysis, DNA; D014162:Transfection