Evaluation of plasmid permanence in transfected cells after transplantation into the rat brain.
Plasmid retention after long-term transplantation has been one of the major technical limitations for transplantation studies. This study describes the use of a modified protocol of Hirt and a SYBR Green-based quantitative real-time PCR (qPCR) to recover and quantify a vector containing a specific transgene in transfected cells after brain transplantation. We compared various methods for sample processing and recovery of extrachromosomal DNA suitable for qPCR. The modified protocol of Hirt was the most reliable for optimal plasmid recovery from transplanted tissue with minimal loss of plasmid DNA compared to a commercial kit or TRIzol(®) protocols. The PCR protocol for plasmid and transgene detection included the design of two highly specific primer sets to detect the sequence for the human glutamate decarboxylase 1 (hGAD(67)) transgene by SYBR Green-based qPCR, and to confirm the presence of vector pREP10 hGAD(67) by end-point PCR. We used a standard curve constructed from serial dilutions of pure plasmid pREP10 hGAD(67) as reference in qPCR experiments to determine the number of plasmid copies recovered from cultured cells and tissue samples after Hirt extraction. Then, plasmid permanence was evaluated in transplanted tissues after different time intervals, and plasmid loss in the tissue of interest was found to be time dependent. In this study we describe an easy, highly specific, low-cost, and reliable method for plasmid recovery and quantification of a transgene of interest in long-term brain transplantation studies; use of this method may be extended to other transplantation models.
Departamento de Neurobiología Conductual y Cognitiva, Instituto de Neurobiología, Universidad Nacional Autónoma de México, Blvd. Juriquilla 3001, Querétaro, Qro. 76230, Mexico.
Jorge H
JH
Limón-Pacheco
Jana
J
Mejía-Toiber
Adriana
A
Gonzalez-Gallardo
Magda
M
Giordano
D000818:Animals; D001483:Base Sequence; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors / Q000302:isolation & purification*; D005968:Glutamate Decarboxylase / Q000235:genetics; D006624:Hippocampus / Q000601:surgery*; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D010957:Plasmids / Q000302:isolation & purification*; D051381:Rats; D017207:Rats, Sprague-Dawley; D060888:Real-Time Polymerase Chain Reaction; D014162:Transfection; D019076:Transgenes
D005968:Glutamate Decarboxylase; C402565:glutamate decarboxylase 1