ABSTRACT
A double-copy Moloney leukemia virus-based retroviral vector containing both the Neo~(R) gene and a mutant human dihydrofolate re-ductase(S31 mutation) cDNA was packaged into the Amphotropic packaging cell hne GP-EAM12( AM12), and a Amphotropic producer cell hne (named AM12-S31)was obtained. In this study, we investigated its drug resistant characteristics, viral titer and for murine hematopoietic progenitor cells transduction as well. MTT assay verified that the AM12-S31 cells were resistant to G418 and methotrexate(MTX), the IC50 were more than 800 ?g/ml and 100 ?M respectively while the control cell line AM12 was sensitive to both drugs, the IC50 were 180 ?g/ml and 10 ?M, respectively. The viral titer for this cell line was approximately 7.8? 104~4.2? 105 G418-resistant colony forming units/ml. The replication-competent virus can not be detected in this producer cell line. We also use the AM12-S31 cells to transfect murine hematopoietic cells (By coculture) . The positive colonies were found in all the G418 concentrations using CFU-GM assay. No G418-resistant colony was found using AM12 transfection. The infected murine marrow cells were returned to lethally irradiated(900rad)recipients. The murine transplanted with AM12-S31 infected marrow cells showed protection from lethal MTX toxicity as compared with AM12 infected animals. Evidence for integration and the proviral DNA was obtained by PCR amplification of proviral DNA. These results indicated this producer cell hne could produce high titer, high-efficiency and non-replcational competent virus. The murine marrow cells could be transfected successfully using this system, and express the foreign gene. The lethal irradiated murine marrow function could be reinstitution by infusing the hematopoietic progenitor cells tranducted with human mutant dihydrofolate reductase. In my opinion, this system would play an important role in research the long-term protection of murine marrow hematopoietic function and drug resistant gene therapy.
ABSTRACT
Objective: To construct TCR V? nucleic acid vaccine. Methods: A fragment of TCR V? gene was amplified by RT- PCR from a human T lymphoma cell line, Jurkat, and cloned into eukaryotic expression plasmid pcDNA3 by gene recombination. After sequencing, the recombinant plasmid was transfected into SP2/0 cells and its expression was detected on mRNA level. BALB/c mice were immunized with pcDNA3 and the recombinant plasmid by intramuscular routes. Antiserum was collected and detected by immunocytochemistry method. Results: A fragment of TCR V? gene from Jurkat cells was obtained and proved to be identical to published sequence. A recombinant plasmid pcDNA3-TCR V? was comstructed. Its expression was detectable on mR- NA level after being transfected into SP2/0 cells. Antiserum from mice immunized with pcDNA3-TCR V? reacted strongly with Ju- rkat cells and SP2/0 cells transfected by pcDNA3-TCR V?, while shows no reaction on CEM cells expressing TCR?? and SP2/0 cells. Antisera from normal force and mice immunized with pcDNA3 were both negative on Jurkat cells. The results of immunocy- tochemistry indicated that BALB/c mice immunized with pcDNA3-TCR V? produced specific antibody to Jurkat TCR V?.Conclu sion: The TCR V? nucleic acid vaccine we constructed can induce humoral immunity.
ABSTRACT
We used retrovial vector LXSN to construct recombinant retroviral vector with mutant membrane bound TNF - ? gene. The vectors were introduced into packaging cell line, CRIP cells. The G418-resistant colonies were selected and the supernatants of the colonies were used to determine the virus titers. The titer of virus was 1? 104CFU/ml and the retroviral vectors were used to tranduced the human gastric cancer cell line, MGC-803 cells. The results of southern blot assay showed thai the targel gene had integrated into the genomic DNA of MGC-803T. MGC - 803Tcells were ablc to kill L929 cell line, but the parent cell line showed no cytotoxicily to the cells at all. There was no any variance in the morphological appearance and growlh rate in vilro of MGC-803N and MGC-803T cells. The results of inoculation in nude mice with these cells indicated that MGC-803T cells showed a considerable decrease in size of tumor. These results suggested that the retroviral vectors expressing mulant TNF -? gene were successfully construed. MGC - 803T cells showed cytotoxicily slrongly lo L929 cell line in vilro and lumorigenicity .
ABSTRACT
The stability of monoclonal antibody-secreting capacity of three human-h uman hybridomas was studied. It was noticed that there were some differences in the morphology, the size, the number of chromosomes and the secreting-capacity of human-human hybrid cells during expanding culture after cloning or for a long lime culture in vitro. The results showed that the stability of human monoclonal antibody-secreting was concerned with loss of some chromosomes or overgrowth of non-secreting population. The number of tetraploid cells was gradually increased after reselection using HAT medium to these different hybridomas. Meanwhile, some of cells which lost certain chromsomes died quickly. The capacity of monoclonal antibody secreting was recovered in some cell lines. It is important to clone the hybrid cells in time or reselect by using HAT medium when expanding the cloned cells.