title: A one-step cloning method for the construction of somatic cell gene targeting vectors: application to production of human knockout cell lines.
abstract: BACKGROUND
  Gene targeting is a powerful method that can be used for examining the functions of genes. Traditionally, the construction of knockout (KO) vectors requires an amplification step to obtain two homologous, large fragments of genomic DNA. Restriction enzymes that cut at unique recognitions sites and numerous cloning steps are then carried out; this is often a time-consuming and frustrating process.
  
  
  RESULTS
  We have developed a one-step cloning method for the insertion of two arms into a KO vector using exonuclease III. We modified an adeno-associated virus KO shuttle vector (pTK-LoxP-NEO-AAV) to yield pAAV-LIC, which contained two cassettes at the two multiple-cloning sites. The vector was digested with EcoRV to give two fragments. The two homologous arms, which had an overlap of 16 bases with the ends of the vector fragments, were amplified by polymerase chain reaction. After purification, the four fragments were mixed and treated with exonuclease III, then transformed into Escherichia coli to obtain the desired clones. Using this method, we constructed SirT1 and HDAC2 KO vectors, which were used to establish SirT1 KO cells from the colorectal cancer cell line (HCT116) and HDAC2 KO cells from the colorectal cancer cell line (DLD1).
  
  
  CONCLUSIONS
  Our method is a fast, simple, and efficient technique for cloning, and has great potential for high-throughput construction of KO vectors.
authors.affiliation: College of Life Sciences, Wuhan University, Wuhan, Hubei, PR China.
authors.forename: Yi
authors.initials: Y
authors.lastname: Liu
authors.forename: Shangze
authors.initials: S
authors.lastname: Li
authors.forename: Huihui
authors.initials: H
authors.lastname: Zhang
authors.forename: Zurong
authors.initials: Z
authors.lastname: Wan
authors.forename: Xiaodong
authors.initials: X
authors.lastname: Zhang
authors.forename: Runlei
authors.initials: R
authors.lastname: Du
mesh_terms: D003001:Cloning, Molecular / Q000379:methods*; D000229:Dependovirus / Q000235:genetics; D004926:Escherichia coli / Q000378:metabolism; D005090:Exodeoxyribonucleases / Q000235:genetics / Q000378:metabolism; D055786:Gene Knockout Techniques; D018390:Gene Targeting; D005822:Genetic Vectors / Q000235:genetics* / Q000378:metabolism; D045325:HCT116 Cells; D057809:HEK293 Cells; D056464:Histone Deacetylase 2 / Q000172:deficiency / Q000235:genetics / Q000378:metabolism; D006801:Humans; D056564:Sirtuin 1 / Q000172:deficiency / Q000235:genetics / Q000378:metabolism
chemical_list: D005090:Exodeoxyribonucleases; C020133:exodeoxyribonuclease III; D056564:Sirtuin 1; D056464:Histone Deacetylase 2