A bicistronic retroviral vector to introduce drug resistance genes into human umbilical cord blood CD34+ cells to improve combination chemotherapy tolerance.

Abstract:

OBJECTIVE:To study whether human umbilical cord blood CD34+ cells transduced with human aldehyde dehydrogenase class-1 (ALDH-1) and multidrug resistance gene (MDR1) have increases resistance to 4-Hydroperoxycyclo-phosphamide (4-HC) and P-glycoprotein effluxed drugs. METHODS:A bicistronic retroviral vector G1Na-ALDH1-IRES-MDR1 was constructed and used to transfect the packaging cell lines GP + E86 and PA317 by LipofectAMINE method, using the medium containing VCR and 4-HC agents for cloning selection and ping-ponging supernatant infection between the ecotropic producer clone and the amphotropic producer clone, we obtained high titer amphotropic 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 retrovirus containing human ALDH-1 and MDR1cDNA under the stimulation of hemopoietic growth factors. RESULTS:Bicistronic retroviral vector construction was verified by restriction endonuclease analysis. Polymerase chain reaction (PCR), reverse transcription (RT)-PCR, Southern blot, Northern blot, fluorescenceactivated cell sorting (FACS) method and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) analyses showed that dual drug resistance genes have been integrated into the genomic DNA of cord blood CD34+ cells and expressed efficiently. The transgenes recipient cells confered 4-fold stronger resistance to 4-HC and 5.5 to 7.2-fold P-glycoprotein effluxed drug than untransduced cells. CONCLUSION:The bicistronic retroviral vector-mediated transfer of two different types of drug resistance genes into human cord blood CD34+ cells and co-expression provided an experimental foundation for improving combination chemotherapy tolerance in tumor clinical trial.

journal_name

Chin Med J (Engl)

journal_title

Chinese medical journal

authors

Wang J,Chen Z,Xia X,Lu D,Xue J,Ruan C

keywords:

subject

Has Abstract

pub_date

2001-01-01 00:00:00

pages

25-9

issue

1

eissn

0366-6999

issn

2542-5641

journal_volume

114

pub_type

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