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为克服逆转录病毒中内部启动子对插入外源基因表达的干扰，将人Ｇ－ＣＳＦ（粒细胞集落刺激因子）ｃＤＮＡ插入双拷贝载体Ｎ２Ａ的３＇ＬＴＲＵ３区上的多克隆位点，酶切鉴定后将重组质粒用电击转导法转入Ｐｓｉ－２嗜单性包装细胞系，经Ｇ－４１８选择压力，两周后挑出阳性克隆，测定感染滴度后转染ＮＩＨ３Ｔ３细胞，选择挑出抗性克隆，测定Ｇ－ＣＳＦ表达量，最高达５３．３Ｕ／１０６细胞。Ｓｏｕｔｈｅｒｎ印迹和Ｎｏｒｔｈｅｒｎ印迹分析证明嵌合的目的基因在感染细胞中通过基因复制而转染到５＇ＬＴＲ的转录单位之外并且在染色体ＤＮＡ中稳定整合，复制产生两个拷贝，在３＇ＬＴＲ和５＇ＬＴＲ中各有一个基因。
To overcome the interference of retroviral internal promoters with the insertion of foreign genes, human G-CSF (granulocyte colony-stimulating factor) cDNA was inserted into the multiple cloning site on the 3’LTRU3 region of the N2A double-stranded vector and digested After identification, the recombinant plasmids were transfected into Psi-2 monocytic packaging cell line by electroporation. After selective pressure of G-418, the positive clones were picked out after two weeks. The titer was determined and transfected into NIH3T3 cells. Resistant clones were tested for G-CSF expression up to 53.3 U / 106 cells. Southern blotting and Northern blot analysis demonstrated that the chimeric gene of interest was transfected into the infected cells by gene replication out of the transcriptional unit of the 5 ’LTR and stably integrated in the chromosomal DNA, duplicating to produce two copies, 3’ LTR and One gene each in 5’LTR.