Functional analysis of gammaretroviral vector transduction by quantitative PCR.
BACKGROUND
In a clinical setting of gene therapy, quantitative methods are required to determine recombinant viral titres and transgene mRNA expression, avoiding the use of reporter genes.


METHODS
We describe procedures based on quantitative polymerase chain reaction (qPCR) designed to assess functional titres of murine leukaemia virus (MLV) vectors, determine proviral copy numbers in transduced cells, and estimate retroviral transgene expression in both target cell lines and mice with transduced chimeric haematopoiesis.


RESULTS
Compared to EGFP titration, proviral DNA detection by qPCR was more accurate in assessing the number of infective particles in supernatants, such that average viral titres in terms of proviral copies per cell were two-fold higher. Transgene mRNA expression was directly determined from the vectors used without the need for reporter assays. A new parameter, defined here as the 'transcription index' (TI), served to establish the association between transcribed transgenic mRNA and each proviral insertion. The TI represents the potential expression of every vector or insertion in each cell type, and is thus useful as a control parameter for monitoring preclinical or clinical protocols.


CONCLUSIONS
The practical use of qPCR is demonstrated as a valuable alternative to reporter genes for the assessment and surveillance of insertion numbers and transgene expression. In combination with protein expression, this approach should be capable of establishing safer therapeutic gene doses, avoiding the potential side effects of high transduction and expression levels.
Meza
N W
NW
Department of Biochemistry and Molecular Biology IV, Universidad Complutense de Madrid, Madrid, Spain.
Puyet
A
A
Pérez-Benavente
S
S
Quintana-Bustamante
O
O
Diez
A
A
Bueren
J A
JA
Segovia
J C
JC
Bautista
J M
JM
D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D016026:Bone Marrow Transplantation; D002460:Cell Line; D015870:Gene Expression; D017930:Genes, Reporter; D005822:Genetic Vectors; D006367:HeLa Cells; D006801:Humans; D009052:Leukemia Virus, Murine / Q000235:genetics*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D016133:Polymerase Chain Reaction / Q000379:methods; D011533:Proviruses / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D014161:Transduction, Genetic
D012333:RNA, Messenger