Experimental Study On Retrovirus-Mediated Transfer And Expression Of Human MDR1 CDNA

Gene transfer is a technology of transfer and expression of exogenouse gene into target cells with physical or biological methods. The use of retroviruses as vectors has allowed highly efficient and stable introduction of foreign genes into target mammalian cells.In our experiment, PA317 was transfected with a retrovirus vector , pHaMDR1/A containing full —length human MDR1 cDNA by using calcium phosphate precipitation and a producer cell, PA317 —Ha MDR1/A1,was generated with a virus titer of 3:35 × 10~5 CFU/ml. The NIH3T3 and K562 cell lines were infected with the virions produced by PA317— HaMDR1/A1 by means of supernant and cocultivation respectively. The colchicine — selected infectants, NIH3T3/MDR1 and K562/MDR1, were tested. A specific 157bp band was detected in the genomic DNA of both N1H3T3/MDR1 and K562/MDR1 by PCR analysis. A distinct 3. 4b band was found in the genomic DNA of the infectants mentioned above by Soutern hybridization. The results showed that the exogenous MDR1 cDNA has been integreted into the genomic DNA of producer cell line, PA317—HaMDR1/A1. The infectious virus particles generated by PA317 HaMDR1 were able to infect both NEH3T3 (mouse) and K562 (human) cells. The exogenous MDR1 cDNA was stably integrated into the genomes of transfectants. The immunocytochemical stai using MoAb agaist P—gp( P— glycoprotein) was stronger in transfectants than in non— transfectants. A specific 157bp band for MDR1 gene was also detected in cDNA from NIH3T3/ MDR1,but not NIH3T3 using RT— PCR analysis; the intensity of the 157bp band of K562/ MDR1 was as 7 folds strong as that of K562 (internal control, β2— microglobulin) . It suggested that the expression of the exogenous mdr1 cDNA integrated in the genomic DNA of the transfectants was higher than the parental cells at the level of both mRNA and protein. A specific band of amplified mRNA from mouse cells was detected by RT—PCR using primerse designed according to the sequences of human MDR1 cDNA. It indicated that the exogenous or inserted human MDR1 cDNA contributed to the overexpression of MDR1 mRNA. The sequences of the primers used in the expriment were compared with that of the mouse mdr1 cDNA.The highest homogeneous rate was 45,% in sense strand and 80% in antisense strand. It was theoretically proven that introdution of exogenous MDR1 cDNA resulted in overexpression of MDR1 mRNA. The drug resistant pattern of K562/MDR1, included resistance to adriamycin (7.8fold),daunorubicine(8.1 fold),mitoxantron(7.0 fold), VP—16(2. 24 fold), and vincristine(2.27fold HHT(4.86 fold).The transfectant did not exhibit resistant to cytarabine. There was no difference of morphology and growth curves between transfectants and nontransfectants. It concluded that an amphotropic virus producer cell line, PA317 —HaMDR1/A1, was established, which possesed the ability to transfect a variety of cell types. The transfectants stably integrated and expressed the exogenous MDR1 cDNA with higher level of MDR1 mRNA and P—gp, without change of biological features. The producer cell line, PA317—HaMDR1/A1 is useful for...