A comparative evaluation of gene transfer into blood cells using the same retroviral backbone for independent expression of the EGFP and deltaLNGFR marker genes.
OBJECTIVE
Retroviral vectors are widely used to deliver foreign genes to hematopoietic stem cells (HSC). Improvement of marking protocols needs reporter genes to allow rapid detection and efficient selection of transduced cells. The great potential of EGFP and LNGFR as reporter systems prompted us to compare them simultaneously, using the same retroviral backbone and the same gene transfer procedures.


METHODS
The EGFP and LNGFR coding sequences were separately cloned into the MFG retroviral backbone. A cloning strategy assuring that both genes utilize the same ATG as the start codon was adopted. Marker gene expression, viral titers, transduction efficiency, and vector stability were evaluated in expanded amphotropic packaging clones and human hematopoietic cell lines by flow cytometry and PCR analysis. Vectors were also tested for their ability to transduce CD34+ peripheral blood cells.


RESULTS
A significantly larger number of MFG- LNGFR packaging clones were obtained that produced high viral titers. A direct correlation between viral titer and marker gene expression in packaging clones was demonstrated for both constructs. Similar expression kinetics and absence of in vitro toxicity in transduced cells were also observed for both constructs. Successful infection of CD34+ cells was achieved even after a short time of exposure to recombinant viruses.


CONCLUSIONS
Our results demonstrate that EGFP and LNGFR marker genes are equally useful for a rapid, specific and non-toxic detection of transduced cells. The MFG-EGFP construct appears useful to optimize gene transfer protocols in vitro. On the other hand, the MFG-LNGFR construct, for making possible a more efficient selection of high titer producer clones, as well as for safety and adaptability to the in vivo use, is more suitable for clinical applications.
Giaretta
I
I
Department of Histology, Microbiology, and Medical Biotechnologies, University of Padova, Italy.
Madeo
D
D
Bonaguro
R
R
Cappellari
A
A
Rodeghiero
F
F
Giorgio
P
P
D002460:Cell Line; D005069:Evaluation Studies as Topic; D005434:Flow Cytometry; D005456:Fluorescent Dyes; D018014:Gene Transfer Techniques / Q000592:standards*; D017930:Genes, Reporter; D005819:Genetic Markers; D005822:Genetic Vectors / Q000138:chemical synthesis; D049452:Green Fluorescent Proteins; D006412:Hematopoietic Stem Cells / Q000821:virology; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics*; D011726:Pyridinium Compounds; D020800:Receptor, Nerve Growth Factor / Q000235:genetics*; D012190:Retroviridae / Q000235:genetics
D005456:Fluorescent Dyes; D005819:Genetic Markers; D008164:Luminescent Proteins; C406028:MitoFlour green; D011726:Pyridinium Compounds; D020800:Receptor, Nerve Growth Factor; D049452:Green Fluorescent Proteins