Objective: To establish an efficient and safe gene transfer system mediated by retrovirus for gene marking and gene therapy of human leukemia. Method: The retroviral vector LXSN, containing the neomycin resistance (Ne...Objective: To establish an efficient and safe gene transfer system mediated by retrovirus for gene marking and gene therapy of human leukemia. Method: The retroviral vector LXSN, containing the neomycin resistance (NeoR) gene, was transferred into amphotropic packaging cells GP+envAm12 by liposome transfection or by ecotropic retrovirus transduction. Amphotropic retrovirus in supernatants with higher titer was used to infect human leukemic cell lines NB4, U937, and THP-1. The efficiency of gene transfer was assayed on colonies formed by transduced K562 cells. Results: The titer of DOSPER directly transfected GP+envAm12 cells determined on NIH3T3 cells was 8.0×105 CFU/ml, while that of producer infected with retrovirus was 1.6×107 CFU/ml. Integration of NeoR gene into all leukemia cells was confirmed by polymerase chain reaction (PCR). Absence of replication-competent virus was proved by both nested PCR for env gene and marker gene rescue assay. Gene transfer with the efficiency as high as 93.3 to 100% in K562 cells was verified by seminested PCR for integrated NeoR gene on colonies after 7 days’ culture. Conclusion: The efficiency and safety of retrovirus mediated gene transfer system might provide an optimal system in gene therapy for leukemia or genetic diseases.展开更多
Interleukin 2 (IL- 2) is a T cell growth factor. In the present study, human IL- 2 cDNA was cloned from total RNA of activated tonsillar mononuclear cells (TMNC) following reverse transcription and PCR amplification u...Interleukin 2 (IL- 2) is a T cell growth factor. In the present study, human IL- 2 cDNA was cloned from total RNA of activated tonsillar mononuclear cells (TMNC) following reverse transcription and PCR amplification using a pair of synthesized primers. DNA sequence analysis demonstrated that the IL- 2 cDNA cloned from the Chinese tonsil donor is identical with the data reported so far, reflecting the high structural conservation of this gene. The human IL- 2 cDNA was inserted into XM6 to construct a recombinant retroviral expression vector XM6-IL2, having human IL-2 cDNA driven by the 5' LTR of MMLV. This vector was successfully passaged through ψ 2 and PA317 cells to yield high producer lines of ecotropic and amphotroplc infectious viruses. The murine myeloma cell line SP2/0 after being infected by retrovirus released from high titer PA317 constitutlvely secreted IL- 2 activity into the culture medium when assayed for T cell proliferative capacity. Activated human T cells are also exposed to the infectious retrovirus XM6- IL2. In sharp contrast to the parallel controls, the infected T cells with or without the furtheraddition of 500 units/ ml of exogenous IL- 2 proliferated and formed colonies of significant size under the selection pressure of G418. However, their growth in vitro could only be maintained for about 3 weeks. These facts demonstrated that gene transfer of human IL- 2 cDNA viaretrovirus is far from being sufficient to maintain the relatively long-termgrowth and clonal expansion of human T cell subpopulations in vitro.展开更多
文摘Objective: To establish an efficient and safe gene transfer system mediated by retrovirus for gene marking and gene therapy of human leukemia. Method: The retroviral vector LXSN, containing the neomycin resistance (NeoR) gene, was transferred into amphotropic packaging cells GP+envAm12 by liposome transfection or by ecotropic retrovirus transduction. Amphotropic retrovirus in supernatants with higher titer was used to infect human leukemic cell lines NB4, U937, and THP-1. The efficiency of gene transfer was assayed on colonies formed by transduced K562 cells. Results: The titer of DOSPER directly transfected GP+envAm12 cells determined on NIH3T3 cells was 8.0×105 CFU/ml, while that of producer infected with retrovirus was 1.6×107 CFU/ml. Integration of NeoR gene into all leukemia cells was confirmed by polymerase chain reaction (PCR). Absence of replication-competent virus was proved by both nested PCR for env gene and marker gene rescue assay. Gene transfer with the efficiency as high as 93.3 to 100% in K562 cells was verified by seminested PCR for integrated NeoR gene on colonies after 7 days’ culture. Conclusion: The efficiency and safety of retrovirus mediated gene transfer system might provide an optimal system in gene therapy for leukemia or genetic diseases.
文摘Interleukin 2 (IL- 2) is a T cell growth factor. In the present study, human IL- 2 cDNA was cloned from total RNA of activated tonsillar mononuclear cells (TMNC) following reverse transcription and PCR amplification using a pair of synthesized primers. DNA sequence analysis demonstrated that the IL- 2 cDNA cloned from the Chinese tonsil donor is identical with the data reported so far, reflecting the high structural conservation of this gene. The human IL- 2 cDNA was inserted into XM6 to construct a recombinant retroviral expression vector XM6-IL2, having human IL-2 cDNA driven by the 5' LTR of MMLV. This vector was successfully passaged through ψ 2 and PA317 cells to yield high producer lines of ecotropic and amphotroplc infectious viruses. The murine myeloma cell line SP2/0 after being infected by retrovirus released from high titer PA317 constitutlvely secreted IL- 2 activity into the culture medium when assayed for T cell proliferative capacity. Activated human T cells are also exposed to the infectious retrovirus XM6- IL2. In sharp contrast to the parallel controls, the infected T cells with or without the furtheraddition of 500 units/ ml of exogenous IL- 2 proliferated and formed colonies of significant size under the selection pressure of G418. However, their growth in vitro could only be maintained for about 3 weeks. These facts demonstrated that gene transfer of human IL- 2 cDNA viaretrovirus is far from being sufficient to maintain the relatively long-termgrowth and clonal expansion of human T cell subpopulations in vitro.
