Hwang L H, Gilboa E
J Virol. 1984 May;50(2):417-24. doi: 10.1128/JVI.50.2.417-424.1984.
Calcium phosphate-mediated DNA transfection and retroviral infection are two alternative gene transfer techniques designed to introduce specific DNA fragments into the chromosomes of recipient cells. To compare the efficiency of expression of genes introduced into cells by either of these two techniques, a retrovirus-derived vector was constructed from the genome of Moloney murine leukemia virus by replacing the coding sequences of the envelope gene with the bacterial Neor gene derived from Tn5, termed rEnv-Neor. Expression of the hybrid Neor gene in NIH 3T3 cells after DNA transfection or retroviral infection was determined by measuring the steady-state levels of the corresponding cytoplasmic polyadeylated RNA species. Cells containing one copy of the integrated rEnv-Neor DNA introduced into cells by retroviral infection expressed 10- to 50-fold-higher levels of vector-specific RNA compared with cells harboring one copy of the same DNA derived by DNA transfection. Analysis of the integrated rEnv-Neor DNA with the methylation-sensitive restriction enzyme SmaI has shown that DNA integrated after DNA transfection but not after viral infection is partially methylated, predominantly in the 5' long terminal repeat, the region involved in initiation of transcription.
磷酸钙介导的DNA转染和逆转录病毒感染是两种不同的基因转移技术，旨在将特定的DNA片段导入受体细胞的染色体中。为了比较通过这两种技术之一导入细胞的基因的表达效率，构建了一种源自莫洛尼鼠白血病病毒基因组的逆转录病毒载体，通过用源自Tn5的细菌新霉素抗性基因（Neor）取代包膜基因的编码序列，称为rEnv-Neor。通过测量相应细胞质多聚腺苷酸化RNA种类的稳态水平，确定DNA转染或逆转录病毒感染后NIH 3T3细胞中杂交Neor基因的表达。与含有通过DNA转染获得的一份相同DNA的细胞相比，通过逆转录病毒感染导入细胞的含有一份整合的rEnv-Neor DNA的细胞表达的载体特异性RNA水平高10至50倍。用甲基化敏感的限制性内切酶SmaI对整合的rEnv-Neor DNA进行分析表明，DNA转染后整合的DNA而不是病毒感染后整合的DNA部分甲基化，主要在5'长末端重复序列中，该区域参与转录起始。
