An efficient approach for constructing shRNA expression vectors based on short oligonucleotide synthesis.
Traditional strategies for establishing shRNA expression constructs are inefficient, error-prone, or costly. We describe a simple approach that overcomes these drawbacks. Briefly, the sense and antisense strands of the short hairpin RNA coding sequence are segmented into two parts, respectively, at asymmetric sites. The four resulting short oligonucleotides are synthesized. Each oligonucleotide is annealed with its opposite, resulting in a double-stranded fragment with sticky termini at both ends. The two fragments so generated can be easily spliced by simple ligation to reconstitute the full-length short hairpin RNA coding sequence which can then be cloned into an appropriately restricted vector.
Li
Xiao-Xia
XX
Key Lab of Ministry of Health for Hormone and Development, Institute of Endocrinology, Tianjin Medical University, Tianjin 300070, People's Republic of China.
Jia
Hong-Wei
HW
Quan
Jin-Xing
JX
Jiao
Yan-Li
YL
Yang
Yu-Hong
YH
Wang
Bao-Li
BL
Yao
Zhi
Z
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D005786:Gene Expression Regulation; D005822:Genetic Vectors / Q000235:genetics*; D037181:Lectins, C-Type / Q000235:genetics / Q000378:metabolism; D008967:Molecular Biology / Q000379:methods*; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000096:biosynthesis*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics*; D051381:Rats; D015183:Restriction Mapping
D037181:Lectins, C-Type; D009841:Oligonucleotides; D012333:RNA, Messenger; D034741:RNA, Small Interfering