Downregulation of human Cdc6 protein using a lentivirus RNA interference expression vector.
Eukaryotic CDC6 gene function is required for the initiation of DNA replication and is a key regulatory protein during cell cycle progression. The human CDC6 gene is not expressed in most normal tissues, in contrast with its marked expression in proliferating cancer cells. An effective way to explore the gene functions of CDC6 is to knock-down the CDC6 messenger RNA (mRNA) and examine the phenotypic consequences. In this chapter, we describe the construction of a lentivirus vector to express a CDC6 DNA segment. The transcript is able to fold by itself because the sense and antisense regions are complementary. There is a 9-nucleotide (nt) loop region allowing for the short hairpin RNA (shRNA) to form. Cellular ribonucleases process the shRNA into a functional short interfering RNA (siRNA). Down-regulation of Cdc6 protein is confirmed by Western blots.
Luo
Feng
F
Division of Hematology-Oncology, University of Southern California and Children's Hospital, Los Angeles, CA, USA.
Yee
Jiing-Kuan
JK
Huang
Sheng-He
SH
Wu
Ling-Tao
LT
Jong
Ambrose Y
AY
D018797:Cell Cycle Proteins / Q000235:genetics*; D002460:Cell Line; D045744:Cell Line, Tumor; D015536:Down-Regulation; D005095:Exoribonucleases / Q000235:genetics; D005822:Genetic Vectors; D006367:HeLa Cells; D006801:Humans; D016086:Lentivirus / Q000235:genetics; D035683:MicroRNAs / Q000235:genetics*; D009687:Nuclear Proteins / Q000235:genetics*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D014161:Transduction, Genetic
C103706:CDC6 protein, human; D018797:Cell Cycle Proteins; D035683:MicroRNAs; D009687:Nuclear Proteins; D034741:RNA, Small Interfering; D005095:Exoribonucleases