Js. Wang et al., A bicistronic retroviral vector to introduce drug resistance genes into human umbilical cord blood CD34(+) cells to improve combination chemotherapy tolerance, CHIN MED J, 114(1), 2001, pp. 25-29
Objective To study whether human umbilical cord blood CD34(+) cells transdu
ced with human aldehyde dehydrogenase class-1 (ALDH-1) and multidrug resist
ance gene (MDR1) have increases resistance to 4-Hydroperoxycyclo-phosphamid
e (4-HC) and P-glycoprotein effluxed drugs.
Methods A bicistronic retroviral vector G1Na-ALDH1-IRES-MDR1 was constructe
d and used to transfect the packaging cell lines GP + E86 and PA317 by Lipo
fectAMINE method, using the medium containing VCR and 4-HC agents for cloni
ng selection and ping-ponging supernatant infection between the ecotropic p
roducer clone and the amphotropic producer clone, we obtained high titer am
photropic PA317 producing cells with high titers up to 5.6 x 10(5) CFU/ml.
Cord blood CD34+ cells were transfected repeatedly with supernatant of retr
ovirus containing human ALDH-1 and MDR1cDNA under the stimulation of hemopo
ietic growth factors.
Results Bicistronic retroviral vector construction was verified by restrict
ion endonuclease analysis. Polymerase chain reaction (PCR), reverse transcr
iption (RT)-PCR, Southern blot, Northern blot, fluorescenceactivated cell s
orting (FACS) method and 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazol
ium bromide (MTT) analyses showed that dual drug resistance genes have been
integrated into the genomic DNA of cord blood CD34+ cells and expressed ef
ficiently. The transgenes recipient cells confered 4-fold stronger resistan
ce to 4-HC and 5.5 to 7.2-fold P-glycoprotein effluxed drug than untransduc
Conclusion The bicistronic retroviral vector-mediated transfer of two diffe
rent types of drug resistance genes into human cord blood CD34(+) cells and
co-expression provided an experimental foundation for improving combinatio
n chemotherapy tolerance in tumor clinical trial.