College of Veterinary Medicine, Northwest A&F University, Yangling 712100, Shaanxi, People's Republic of China.
Biotechnol Lett. 2012 Jul;34(7):1251-5. doi: 10.1007/s10529-012-0912-9. Epub 2012 Apr 3.
cDNA is widely used in gene function elucidation and/or transgenics research but often suitable tissues or cells from which to isolate mRNA for reverse transcription are unavailable. Here, an alternative method for cDNA cloning is described and tested by cloning the cDNA of human LALBA (human alpha-lactalbumin) from genomic DNA. First, genomic DNA containing all of the coding exons was cloned from human peripheral blood and inserted into a eukaryotic expression vector. Next, by delivering the plasmids into either 293T or fibroblast cells, surrogate cells were constructed. Finally, the total RNA was extracted from the surrogate cells and cDNA was obtained by RT-PCR. The human LALBA cDNA that was obtained was compared with the corresponding mRNA published in GenBank. The comparison showed that the two sequences were identical. The novel method for cDNA cloning from surrogate eukaryotic cells described here uses well-established techniques that are feasible and simple to use. We anticipate that this alternative method will have widespread applications.
cDNA 广泛应用于基因功能阐明和/或转基因研究，但通常难以获得用于反转录的合适组织或细胞。本文描述了一种 cDNA 克隆的替代方法，并通过从基因组 DNA 克隆人 LALBA（人乳白蛋白）的 cDNA 进行了验证。首先，从人外周血中克隆包含所有编码外显子的基因组 DNA，并将其插入真核表达载体。接下来，通过将质粒导入 293T 或成纤维细胞，构建替代细胞。最后，从替代细胞中提取总 RNA，并通过 RT-PCR 获得 cDNA。获得的人 LALBA cDNA 与 GenBank 中公布的相应 mRNA 进行比较。比较结果表明，两条序列完全一致。本文描述的这种从替代真核细胞中克隆 cDNA 的新方法使用了成熟的技术，具有可行性和易用性。我们预计这种替代方法将具有广泛的应用。
