Toneguzzo F, Keating A, Glynn S, McDonald K
EG & G Biomolecular, Natick, MA 01760.
Nucleic Acids Res. 1988 Jun 24;16(12):5515-32. doi: 10.1093/nar/16.12.5515.
Southern analysis of individual transfectants generated by electroporation demonstrated a strong preference for the integration of DNA in low copy number even when electroporation was performed in the presence of increasing DNA concentrations. Although transfer of multiple DNA copies was detected at higher DNA concentrations (16 pmoles/ml or greater), the average gene copy number even at 36 pmoles DNA per ml, was only 13. Multiple gene copies were integrated at either a few chromosomal sites, or at a single site within individual transfectants. Restriction endonuclease cleavage data were consistent with a random orientation of molecules within a concatemer, suggesting that the concatemer may have risen via end-to-end ligation of linear molecules, rather than by homologous recombination. Integration of exogenous DNA into the host chromosome occurred preferentially at the ends of the linear molecule. Although the linearization site was lost upon integration, endonuclease sites as close as 18 bp from the linearization site were retained. These data, as well as direct restriction mapping of the transferred genes, indicate that DNA transfer and integration occur without DNA rearrangement. Taken together, these results suggest that electroporation may offer some unique advantages for the transfer of eukaryotic genes.
对通过电穿孔产生的单个转染子进行Southern分析表明，即使在DNA浓度不断增加的情况下进行电穿孔，DNA也强烈倾向于以低拷贝数整合。尽管在较高DNA浓度（16皮摩尔/毫升或更高）下检测到多个DNA拷贝的转移，但即使在每毫升36皮摩尔DNA的情况下，平均基因拷贝数也只有13个。多个基因拷贝整合在少数几个染色体位点，或单个转染子内的单个位点。限制性内切酶切割数据与串联体中分子的随机取向一致，表明串联体可能是通过线性分子的端对端连接形成的，而不是通过同源重组。外源DNA优先整合到宿主染色体的线性分子末端。尽管整合时线性化位点丢失了，但距线性化位点仅18个碱基对的内切酶位点得以保留。这些数据以及转移基因的直接限制性图谱表明，DNA转移和整合过程中没有DNA重排。综上所述，这些结果表明电穿孔可能为真核基因的转移提供一些独特的优势。
