title: An alternative method for cDNA cloning from surrogate eukaryotic cells transfected with the corresponding genomic DNA.
abstract: 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.
authors.affiliation: College of Veterinary Medicine, Northwest A&F University, Yangling 712100, Shaanxi, People's Republic of China.
authors.forename: Lin-Yong
authors.initials: LY
authors.lastname: Hu
authors.forename: Chen-Chen
authors.initials: CC
authors.lastname: Cui
authors.forename: Yu-Jie
authors.initials: YJ
authors.lastname: Song
authors.forename: Xiang-Guo
authors.initials: XG
authors.lastname: Wang
authors.forename: Ya-Ping
authors.initials: YP
authors.lastname: Jin
authors.forename: Ai-Hua
authors.initials: AH
authors.lastname: Wang
authors.forename: Yong
authors.initials: Y
authors.lastname: Zhang
mesh_terms: D002478:Cells, Cultured; D003001:Cloning, Molecular / Q000379:methods*; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D018076:DNA, Complementary / Q000235:genetics* / Q000378:metabolism; D005822:Genetic Vectors; D006801:Humans; D007768:Lactalbumin / Q000235:genetics; D007962:Leukocytes / Q000201:enzymology; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D018076:DNA, Complementary; D012313:RNA; D004247:DNA; D007768:Lactalbumin