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Malaria is a parasitic disease which threatens human life and health seriously. Sulfadoxine-pyrimethamine (SP) has been recommended for intermittent preventive treatment of malaria in children and pregnant women, and also used as a compound component of artemisinin based therapy. The mechanisms of SP resistance in P. falciparum involve point mutations in the genes encoding dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS), and the drug pressure can also lead to the mutations in the two genes of P. vivax. To provide the information for the formulation of anti-malarial strategies, this article reviews the discovery, application, effect of SP, and the resistance mechanism and research progress of the related genes in P. vivax .

Pneumocystis jirovecii pneumonia is an opportunistic infection among immunocompromised people. Studies have found that the increased resistance to sulfa drugs of Pneumocystis jirovecii may be associated with the mutation of dihydropteroate synthase ( DHPS ) gene and dihydrofolate reductase ( DHFR ) genes, but the mechanism is still unclear. The mutation of DHPS and DHFR genes may be the result of sulfa drugs selection or spontaneous genetic polymorphism, and it can be acquired from person-to-person transmission. This article reviews the cause, molecular epidemiology of DHPS and DHFR gene mutation, and the relationship between DHPS and DHFR gene mutation and clinical outcomes.

Objective: To investigate the effects of dihydrofolate reductase (DHFR) gene expression up-regulating on cisplatin resistance in epithelial ovarian cancer cell lines., Methods: The cDNA length of DHFR gene was amplified by PCR and was connected to lentiviral vector pWPI, the recombinant retroviral vector DHFR-pWPI was infected SKOV3 cells by lipofectamine 2000. The groups included DHFR-pWPI-SKOV3 cell, pWPI-SKOV3 cell and SKOV3 cell group. Western blot was used to detect the expression of DHFR. Flow cytometry was applied to measure the cell apoptosis rate of 3 groups cells in different cisplatin concentrations (2.5, 5.0, 10.0, 20.0 µg/ml) and at different time period (24, 48 and 72 hours), and half maximal inhibitory concentration (IC50) treated with cisplatin concentration (6.0, 4.0, 4.9 µg/ml). High performance liquid chromatography (HPLC) was applied to test intracellular cisplatin concentration in different cisplatin concentration (4.0, 6.0, 8.0 µg/ml) at 24 and 48 hours. Transmission electron microscope was used to observe ultrastructural changes cells under IC50 cisplatin concentration., Results: The recombinant plasmid DHFR-pWPI was constructed and then infected into SKOV3 cell successfully. (1) The expression of DHFR detected by western blot in transfection group was higher than those in the negative control group and blank control group (10.280±0.009 vs 2.050±0.003 vs 3.480±0.003; P<0.01). (2) Treated with cisplatin concentration (2.5, 5.0, 10.0, 20.0 µg/ml) at 24, 48 hours, the apoptosis rate detected by flow cytometry results shown that they were lower than those in the negative control group and blank control group (P<0.05), while treated at the concentration of 5.0 and 10.0 µg/ml for 72 hours, whose apoptosis rate in transfection group was higher than those in the negative control group and blank control group (P<0.05). When treated cells under IC50 cisplatin concentration (6.0, 4.0, 4.9 µg/ml) at for 24 and 48 hours, the results indicated that there were mainly G0/G1 stage cell cycle rate in 3 groups, it was obviously higher in transfection group than those in two control groups (P<0.05). However, mainly G2/M, S stage cell cycle rate for 72 hours, and S stage cell cycle rate in transfection group obviously higher than those in two control groups, but G2/M stage cell cycle rate were lower (P<0.01). (3) After treated with cisplatin concentration (4.0 µg/ml) for 24, 48 hours and cisplatin concentration (6.0 µg/ml) for 24 hours, the intracellular cisplatin content tested by HPLC method in the transfection group were significantly lower than those in two control groups (P<0.01). While, at 6.0 µg/ml of cisplatin concentration for 48 hours and 8.0 µg/ml of cisplatin concentration for 24 and 48 hours, the intracellular cisplatin content of transfection group were obviously higher than those two control groups (P<0.05). (4) Treated with IC50 (6.0, 4.0, 4.9 µg/ml) cisplatin concentration at different time to obeserve ultrastructural changes by transmission electron microscopy. The results shown that the microwire gathered together at 24 and 48 hours, and the number and structure of mitochondria had obvious change in transfection group, while there was rare microfilament, the number of mitochondria decreased but structure change was not apparent in two control groups. There were appeared expansion of endoplasmic reticulum and had rare normal organelles among three groups. After treated with cisplatin for 72 hours, there were inordinate microfilament, a part of nuclear membrane disappeared, a lot of ribosomes gathered together in two control groups, and there were rare microfilament in transfection group, nuclear membrane completely disappeared, many white cystic matter were seen in cytoplasm, mitochondrial structure disappeared completely, which seems most cells on the verge of death., Conclusion: The lentiviral expressing vector harboring human DHFR gene were successfully constructed. When the up- expression of DHFR gene, the drug-resistant in ovarian cancer cell may be increase, which suggest that there were certain contact between resistance increases with microfilament gathered and the change of the mitochondria.

Effective chemotherapy is the mainstay of malaria control. However, it is undergoing the serious threat by resis- tance of falciparum malaria to antimalarial drugs. In recent years, with the development of molecular biology technology, molec- ular markers have been widely used to monitor antimalarial drug resistance. This paper reviews the researches on the common molecular markers related to Plasmodiumfalciparum drug resistance.

Stable transformants for mammalian cells from gene transfer often show extreme variability in expression of the introduced transgene. This occurs from the highly variable number of copies integrated into the genome and from position effects on gene expression due to random integration. We constructed engineered CHO strains that can be used for high-level production of foreign proteins by gene-targeting. After transfecting dihydroforate reductase (DHFR)-deficient CHO cells with a newly screening vector plasmid pMCEscan, which carrying a FRT-neo*-IRES-k2tPA fusion gene and a DHFR gene, we screened colonies by k2tPA expression level. We selected 7 clones that expressed high level of k2tPA and carried one copy of the plasmid in their chromosomes. These clones showed in high level k2tPA production without amplification. So we targeted reporter gene (k2tPA) to test the basal expression ability of these cells clones. The clone, 8-1, showed the same effect to high base expression level. In this clone, the FRT-neo*-IRES-tPA gene was integrated at a transcription-active, DHFR-mediated, gene-amplifiable locus in the chromosomes. A gene-targeting vector, carrying a FRT-fused hygromycin-resistance gene, was constructed to target desired genes in chromosomal FRT by FLP recombinase-mediated site-specific recombination. Using this cell-vector system, we could reproducibly obtain high producers of recombinant proteins by gene-targeting and gene amplification. Using the site-specific integration CHO/dhfr- cell line 8-1, the expression level of k2tPA could amount to 17.1 microg/10(6) cell x 24 h.

Objective: To explore the feasibility of transfecting DHFR (human double-mutant dihydrofolate reductase) gene into mouse bone marrow cells and the effect of resistance to high dose MTX chemotherapy., Methods: After DHFR gene was transfected into mouse bone marrow cells with retroviral vector, the cells were treated with methotrexate (MTX) and then CFU-GM (granulocyte-macrophage colony-forming unit) assay was performed. Peripheral blood leucocytes and platelets, body weight and survival rate were observed. After treatment with high dose MTX, the expression of drug resistance gene was checked by RT-PCR in the transfected bone marrow cells., Results: SFG-F/S-NeoR gene-transfected mice bone marrow cells yielded drug-resistance colonies to MTX (donor mice: 15.8%, recipient mice: 18.0%, control: 0) The peripheral blood leucocytes and platelets, body weight recovered gradually and the survival rate was 83.3% at the 40th day, while 0 in controls in gene transfected mice after large dose MTX treatment. RT-PCR of transgenic mouse marrow cells showed the band of F/S gene (400 bp)., Conclusion: DHFR gene can not only be integrated and expressed in bone marrow cells but also improve their drug-resistence to MTX.

Objective: To explore the feasibility of transferring fusion gene of dihydrofolate reductase (DHFR) gene and cytidine deaminase (CD) gene into mouse bone marrow cells in order to observe the drug resistance of high dose methotrexate (MTX) and cytosine arabinoside (Ara-C) in the bone marrow cells and to improve the tolerance of myelosuppression following combination chemotherapy., Methods: Human double-mutant dihydrofolate reductase-cytidine deaminase fusion gene was transferred into two mice bone marrow cells by retroviral vector. Resistant colony-forming unit granulocyte-macrophage (CFU-GM) assays were performed in mouse bone marrow cells by retroviral infection and after treatment by drugs (Ara-C, MTX, and Ara-C + MTX). DNA was extracted from mouse bone marrow cells. The expression of drug resistant genes in mouse bone marrow cells after transferring by retroviral vector was checked by polymerase chain reaction (PCR)., Results: Bone marrow cells after coculture with the retroviral producer cells transduced with the genes (SFG-F/S-CD) showed the drug resistance colonies yield (Colony formation after exposure to Ara-C, MTX and Ara-C + MTX were 56%, 22% and 14%, respectively) and the increase in drug resistant to both MTX and Ara-C (P < 0.005). Expression of DHFR and CD gene in extracted DNA of transfected mice were demonstrated by PCR., Conclusions: Double drug resistant gene can not only integrate and co-express in mice bone marrow cells but also increase the drug resistance to MTX and Ara-C.

In recent years, Bacterial resistance is more and more serious for the irrational use of antibiotics produces resistant strains and other reasons. Human are trying to solve the problem from different ways, including the study of antimicrobial peptides. Defensin is one of the most important of antimicrobial peptides. A novel antimicrobial peptide, human beta-defensin 3, was isolated and demonstrated a salt-insensitive broad spectrum of potent antimicrobial activity against many potentially pathogenic microbes. The total RNA was extracted from human tonsil and the hbetaD-3 specific cDNA sequence was amplified with RT-PCR. After sequenced, the target DNA fragment was cloned into pQE-80L vector together with the DNA fragment encoding carrier protein DHFR. The recombinant vectors were transformed into E. coli M15 and the expression was induced based on the optimal values of the IPTG concentration incubation temperature and induction time determined in the previous section. The expressed proteins were analyzed by SDS-PAGE and Western-blotting. The mass of the recombinant protein was about 40% of total bacteria protein. Isolate and purify the target protein. The recombinant hbetaD-3 fusion proteins possess the antimicrobial activity to staphylococcus aureus, multiresistant staphylococcus aureus (only vancomycin-sensitive) and Candida albicans in the assay of drug susceptibility. Advanced study can be continued based on our experiments.

Gene amplification is a common mechanism that contributes to the drug resistance. To explore the molecular genetic background related to the MTX resistance in the mouse MTX-resistant cells, differential PCR was used to determine the amplification and overexpression of DHFR gene. In addition, the correlations between c-myc, p53 status and dhfr amplification were studied. Amplification and overexpression of dhfr suggested its role in MTX-resistant cells. However, no amplification and overexpression of c-myc were detected. On the other hand, no alteration of p53 copy number was found. The increased mRNA level of p53 suggested the normal function of p53. These results implicated the status of c-myc and p53 had no correlation with dhfr amplification, therefore some other molecular genetic alterations may exist to permit the dhfr amplification in MTX-resistant cells.

Objective: To investigate the expression of human-mouse chimeric antibody ch-BD1 against human bladder cancer and its affinity to human bladder cancer in vitro and in vivo., Methods: Three kinds of mutated fragments of dihydrofolate reductase (DHFR) gene were created by techniques of molecular biology to decrease the transcriptional activities and then cloned into pDHL-BD1 so as to construct the vectors pWSD1-BD1, pWSD2-BD1, and pWSD3-BD1 expressing the human-mouse chimeric antibody ch-BD1 against human bladder cancer with decreased expression of DHFR gene. These vectors and pDHL-BD1 were transfected into Chinese hamster ovary cell (CHO)/DHFR- cell respectively. 72 hours later Northern blotting was used to examine their DHFR gene expression. Methotrexate (MTX) of increasing concentrations was added into the culture of transfected CHO/DHFR-cells. The ch-BD1 levels in the supernatants were measured. Purified ch-BD1 was labeled by (99m)TcO(4)(-) and added into the serially diluted solutions of human bladder cancer EJ cells to examine their radioactivities and calculate their affinity constants. EJ cells were injected into the roots of hind limb of 3 Balb/C mice. Four weeks later, (99m)TcO(4)(-)-labeled antibody ch-BD1 were injected into the rats' caudal veins. Radioimmunoimaging was conducted to examine the distribution of the antibody., Results: The sequence of DHFR gene expression levels from strong to weak in the constructed vectors was as follows: pDHL-BD1 > pWS1-BD1 > pWS3-BD1 > pWS2-BD1. When the concentration of MTX was 10(-6) mol/L the expression level of ch-BD1 was significantly correlated to the expression level of DHFR gene, the lower the baseline expression level of DHFR gene the higher the expression level of ch-BD1. After the serially diluted EJ cells were co-incubated with the (99m)TcO(4)(-)-labeled antibody ch-BD1 the immunoactivity ratio of ch-BD1 was 76%, and that of murine monoclonal antibody was 81%; the affinity constant of ch-BD1 was 3.56 x 10(9) M(-1), and that of murine monoclonal antibody BD1 was 1.22 x 10(9) M(-1). 6 hours after injection of the (99m)TcO(4)(-)-labeled antibody ch-BD1 into the mice body it was mainly distributed in the tumor, 22 hours later, it was specifically distributed in tumor, and 24 hours later it was still concentrated here., Conclusion: Decrease of the baseline expression level of DHFR gene effectively increases the amplification of MTX to exogenous gene. The human-mouse chimeric antibody ch-BD1 shows an ideal affinity activity to human bladder cancer in vivo and in vitro and has a certain clinical prospect.

Aim: To increase the expressed level of a human-mouse chimeric antibody against human bladder tumor in dihydrofolate reductase (DHFR) defective CHO cells(CHO/DHFR) via weakening the transcription of DHFR gene in the vector., Methods: A series of chimeric antibody expression vectors with different deletions and mutations in the modulator sequence of DHFR gene were constructed to downregulate the DHFR gene expression. The vectors were used to transfect CHO/DHFR cells and the transfected cells were subjected to gene amplification in medium containing gradually increasing methotrexate (MTX). The expressed chimeric antibody was quantitated by ELISA., Results: The downregulation of vector-produced DHFR gene introduced by mutation of the modulator sequence could significantly improve the gene amplification effect and the increased antibody production correlated to the reduction of DHFR gene expression. From the best group, a clone with antibody production of 55 microg/(10(6) cells.24 h) was obtained by subcloning. More than 100 microg/(10(6) cells.24 h) was achieved by zinic ion induction., Conclusion: MTX induced-increase of recombinant antibody production in CHO cells can be increased by weakening the expression of DHFR gene.

Background & Objective: Methotrexate has been used to treat malignant tumor for 50 years. Different mechanisms of methotrexate resistance has been known in various kinds of leukemias. Because of individualized chemotherapy according to different drug metabolism in different leukemia patients, the treatment became more reasonable and scientific and the treatment effect has been gradually improved. However, there was few report about its drug resistance mechanism in lymphoma. This study was designed to establish Burkitt's lymphoma methotrexate resistant cell strain--Namlwa 12/MTX and study its resistant mechanism., Methods: Namlwa 12/MTX resistant cell strain was established by repeated impulsed exposure to methotrexate. The difference of the methotrexate effect on Namlwa 12/MTX and Namlwa cell strains were evaluated with MTT method. The level of dihydrofolate reductase (DHFR) mRNA expression was assayed with RT-PCR. The function of the reduced folate carrier (RFC) were tested with 3H-MTX labeling and counted with beta-liquid scintillation counter. The capacity to form the polyglutamate methotrexate (MTXPG) in these cell strains was assayed with 3H-MTX labeling and high pressure liquid chromatography (HPLC) separation., Results: More than 20 times methotrexate resistance was found in Namalwa 12/MTX cell strain in comparison with the Namalwa cell strain. This resistance was not associated with the dysfunction of the RFC, but was closely associated with the amount of MTXPG formed. The amount of total MTXPG (MTXPG1-6) formed in these 2 cell strains was (1583 +/- 26) pmol/10(9) tumor cells and (4453 +/- 236) pmol/10(9) tumor cells, respectively (P < 0.05), while the amount of long chain MTXPG (MTXPG4-6) formed only accounted for 5.5% and 25.4% of the MTXPG1-6, respectively (P < 0.05). The level of DHFR mRNA expression was gradually increased with the formation of the drug resistance in Namalwa 12/MTX., Conclusion: Synthesizing difficulty of MTXPG and over-expression of DHFR mRNA level result in methotrexate resistance in Namalwa 12/MTX cell strain.

Background: To study the effect of gene amplification and selection system with DHFR plus GS and DHFR or GS gene on the foreign gene expression., Methods: Using the N-terminal truncated hTPO(T184) gene as target gene, two plasmidsre were constructed: pDC- T184 and pGC-T184 where DHFR and GS gene were used respectively as the selective amplification marker. They were cotransfected into CHO dhfr cells to establish dual gene amplification and selection system of DHFR plus GS gen and respectively transfected to establish single gene amplification and selection system of DHFR or GS gene. Three selective methods in dual selective system to compare expression efficiency of hTPO were designed: the first method (DG) was to use drug pressure of MTX, then use MSX; the second method (GD) was reversed; the third method was simultaneously to use MTX and MSX as drug pressure., Results: DHFR+GS dual system had not only higher gene amplification efficiency but also higher level expression. There was no distinct affect in different method of drug pressure., Conclusions: MTX plus MSX dual drug pressure in dual selection system was an efficient and simple method to increase the expression of foreign gene in mammalian cells.

Objective: To investigate the chromosome aberrations and carcinogenicity of CHO-dhfr- cell induced by integration of plasmid containing S+ and Pre S1 fusion gene of hepatitis B surface antigen (HBsAg)., Methods: The plasmid pCHBSS1G was constructed with S+ Pre S1 of HBsAg. CHO-dhfr- cells were transformed with this recombinant plasmid DNA and CHO cells lines with integrated DNA were cloned and named GdSS118. The GdSS1 18 cell lines secreting the S+ Pre S1 fusion protein of HBsAg at high level were developed by screening in increased concentration of MTX and MSX in cultured media. To make sample of cells chromosome, the cells integrated DNA were subcutaneously injected into nude mouse., Results: The CHO-dhfr- cell lines integrated with S and Pre S1 fusion protein of HBsAg were developed and named GdSS1-18 cell lines. The frequencies and type of chromosome aberrations of the GdSS1-18 cell lines with different passage generations were 11%, 56% and 29%, respectively, while that of the control CHO-dhfr- cell lines was 6%. There was no change in the mode of chromosomes, both cell lines having 20 chromosome s. Both cell lines were non-oncogenic in nude mouse., Conclusions: The chromosome aberration of CHO-dhfr- cells with integrated DNA were obviously higher than that of the original CHO-dhfr- cells without integrated DNA. Both cell lines were non oncogenic in nude mouse.

Objective: To explore the feasibility of cotransferring human mdr-1 gene and DHFR gene into human CD(34)(+) progenitor cells to broaden the spectrum of drug resistance and improve the tolerance of myelosuppression following combination chemotherapy., Methods: The recombinant retroviral vector pSF-DIM containing mdr-1 and DHFR (L22Y) gene was constructed by introducing IRES sequence into vector FMCF which enable highly efficient gene expression in early hematopoietic cells. The retrovirus titers were raised by repeated supernatant cross infection between the amphotropic and ectropic retroviral packaging cells. Human CD(34)(+) progenitor cells were transduced by supernatant infection. Expression of P-gp was detected by flow cytometry. Integration of the foreign drug resistance gene in CD(34)(+) cells was determined by PCR. Drug resistance was evaluated by CFU-GM assay., Result: Integration of the two foreign drug resistance genes was detected in the CD(34)(+) cells after pSF-DIM transduction. Compared with the untransduced group, the expression of P-gp elevated by 10.98% after gene transduction and the CFU-GM yields were significantly increased at 48 nmol/L of MTX and 10 ng/ml or 12 ng/ml of taxol (P < 0.05)., Conclusion: The retroviral vector pSF-DIM can mediate mdr-1 and DHFR gene integration and co-expression in human hematopoietic progenitor cells so as to broaden the spectrum of drug resistance.

Objective: To set up an efficient expressing system for recombinant hepatitis B virus surface antigen (HBsAg) in dhfr gene negative CHO cell line., Methods: HBsAg gene expressing plasmid pCI-dhfr-S was constructed by integrating HBsAg gene into plasmid pCI which carries dhfr gene. The HBsAg expressing cell line was set up by transfection of plasmid pCI-dhfr-S into dhfr gene negative CHO cell line in the way of lipofectin., Results: Under the selective pressure of MTX, 18 of 28 clonized cell lines expressed HBsAg, 4 of them reached a high titer of 1:32 and protein content 1-3 micrograms/ml., Conclusion: In this study, the high level expression of HBsAg demonstrated that the dhfr negative mammalian cell line when recombined with plasmid harboring the corresponding deleted gene can efficiently express the foreign gene. The further steps toward building optimum conditions of the expressing system and the increase of expressed product are under study.

Objective: To investigate the gene point mutation in the dihydrofolate reductase-thymidylate synthase(dhfr) gene of Plasmodium falciparum isolate from Yunnan Province strongly associated with pyrimethamine and cycloguanil resistance., Methods: Nested PCR and restriction endonuclease digestion were applied to detect the gene mutation using dried blood filter paper collected from the fields in Yunnan Province., Results: Different mutations were found in 4 amino acids at positions 16, 51, 108 and 164 of dhfr gene, particularly, Asn-108 and Ile-51, the mutation frequency being 94.1% and 90.1%, respectively. The frequency of the wild-type genotype (3D7-type) Ser-108 appeared lower (9.1%), while the frequency of the Ala-16 was high(61.8%); the mutation-type was very high, the ratio of HB3-type, 7G8-type/FCR3-type and Cambodian-type was 1:21:7.5., Conclusion: The investigation first demonstrated that Plasmodium falciparum Yunnan isolate dihydrofolate reductase-thymidylate synthase gene (dhfr) at positions 16, 51, 108 and 164 exhibited different degrees of point mutation. The frequency of mutation of the 7D8-type involved in pyrimethamine resistance was higher, while that of the FCR3-type involved in cycloquanil resistance was lower.

Objective: To observe the effect of donor murine bone marrow, which was transfected in vivo with mutant hDHFR gene, on the reconstitution and protection of hematopoiesis of lethally irradiated recipient mice., Methods: Mice bone marrow transduced in vivo with mutant hDHFR gene were used for transplantation. After whole bodily irradiated (8.5-9.0 Gy) and then transplanted with the transduced marrow, the recipients were treated with methotrexate (4 mg/kg twice a week for the first week; and then 10 mg/kg twice a week). The survival rate and blood picture of the recipients as well as the integration and expression of hDHFR gene were studied by using PCR and Southern blotting., Results: The hematopoiesis of the recipient mice could be reconstituted and protected from lethal toxicity of high dose methotrexate. It was confirmed that hDHFR gene was integrated into hematopoietic cell as well as some tissues genomic DNA and expressed successfully in the recipients., Conclusion: Donor mice bone marrow transfected with mutant hDHFR gene can effectively reconstitute and protect hematopoiesis in lethally irradiated recipient mice.

Objective: To examine if human peripheral blood hematopoietic progenitor cells transduced with mutant dihydrofolate reductase(mDHFR) gene may increase their resistance to metrotrexate(MTX)., Methods: After enrichment with a high-gradient magnetic cell sorting system(MACS), the peripheral blood CD34+ cells were transfected with supernatant of retrovirus containing human mDHFR cDNA. Colony formation of hematopoietic progenitor cells was utilized to evaluate mDHFR-conferred resistance to MTX. The number of colonies formed from every 500 cells seeded was counted and expressed as the percentage of colony formation., Results: After the mD-HFR-transduced CD34+ cells had been screened in vitro in medium containing 20 nmol.L-1 MTX for 14 days, the percentage of colony formation of mDHFR cDNA-transduced and of mock-transduced CD34+ cells was 29.4% and 16.3%, respectively (P < 0.01). The resistance to MTX was increased twofold., Conclusion: Transduction of mDHFR gene could confer the resistance of human hematopoietic progenitor cells to MTX toxicity.

Objective: To explore whether human umbilical cord blood hematopoietic progenitor cells transduced with mutant dihydrofolate reductase(mDHFR) gene increase resistance to metrotrexate(MTX)., Methods: Cord blood CD34+ cells were enriched with a high-gradient magnetic cell sorting system(MACS), and then transfected with supernatant of retrovirus containing human mDHFR cDNA. Hematopoietic progenitor clonogenic assay was used to evaluate mDHFR-conferred resistance to MTX., Results: The purity of cord blood CD34+ cells was approximately 90% and recovery rate was 71.1%. The transduced and mock-transduced CD34+ cells were cultured in mediums containing 20 nmol/L MTX for 14 days. The percentages of progenitor colonies of transduced and mock-transduced CD34+ cells were higher than that of non-transduced (P < 0.01), obviously. The resistance of the former to MTX is nearly twofold of the latter., Conclusion: Transduction of mDHFR gene could confer the resistance of human hematopoietic progenitor cells to MTX toxicity.

Ninety-seven Shigella strains from 1982 to 1987 were isolated. When these isolates were tested with minimal inhibitory concentrations of trimethoprim, twenty-two were found to be resistant. Among these, 10 were found to be S. flexneri, and 12 to be S. sonnei. In addition, all of these 22 isolates were found to have multiple drug resistance. In order to determine the type of dihydrofolate reductase (DHFR) genes in these 22 trimethoprim-resistant isolates, colony hybridization with probes representing types I, II, III and V DHFR, were used. The results revealed the presence of type I DHFR gene in all of these isolates. Further southern hybridization indicated that the type I DHFR gene was located on plasmids. In order to determine whether a trimethoprim resistant gene existed as a functional transposable element in these isolates, we used a specific probe to detect the prevalence of Tn7. However, no positive hybridization was found. The relationship between the trimethoprim resistant gene and integron is characterized by the fact that the integrase gene, in our study, was frequently found in all of the 22 resistant isolates. These results indicate that trimethoprim resistant genes might transfer among bacteria via site-specific integration.