PMID- 22484114
OWN - NLM
STAT- MEDLINE
DCOM- 20121126
LR  - 20120723
IS  - 1873-4863 (Electronic)
IS  - 0168-1656 (Linking)
VI  - 160
IP  - 3-4
DP  - 2012 Aug 31
TI  - Construction of a long hairpin RNA expression library using Cre recombinase.
PG  - 129-39
LID - 10.1016/j.jbiotec.2012.03.016 [doi]
AB  - A large number of genome-wide screens using RNA interference (RNAi) libraries 
      have been utilized to determine the function of individual gene products involved 
      in a variety of biological processes. In this study, we describe a new method to 
      enzymatically generate a long hairpin RNA (lhRNA) expression library from a cDNA 
      plasmid library using a nicking endonuclease, BcaBEST DNA polymerase, and Cre 
      recombinase without excising the inserted DNA fragment from the plasmid vector. 
      This method involves 5 steps: (1) conversion of an inserted DNA fragment in a 
      plasmid into a direct repeat (DR); (2) purification of the plasmid containing the 
      DR; (3) subcloning a lox71 cassette into the plasmid; (4) conversion of the DR in 
      the plasmid into an inverted repeat (IR) using Cre recombinase; and (5) 
      purification of the plasmid containing the IR. We also established an efficient 
      method for inserting DNase I-digested DNA fragments into expression plasmids to 
      enable construction of a cDNA plasmid library suitable as source materials to 
      construct the lhRNA expression library. We confirmed that each of the lhRNA 
      expression plasmids constructed using this method induced strong RNAi in a 
      silkworm cell line, NIAS-Bm-oyanagi2.
CI  - Copyright (c) 2012 Elsevier B.V. All rights reserved.
FAU - Tomimoto, Kazuya
AU  - Tomimoto K
AD  - Insect Mimetics Research Unit, National Institute of Agrobiological Sciences, 1-2 
      Owashi, Tsukuba, Ibaraki 305-8634, Japan.
FAU - Yamakawa, Minoru
AU  - Yamakawa M
FAU - Tanaka, Hiromitsu
AU  - Tanaka H
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
DEP - 20120330
PL  - Netherlands
TA  - J Biotechnol
JT  - Journal of biotechnology
JID - 8411927
RN  - 63231-63-0 (RNA)
RN  - EC 2.7.7.- (Cre recombinase)
RN  - EC 2.7.7.- (Integrases)
SB  - IM
MH  - Cloning, Molecular/*methods
MH  - *Gene Library
MH  - Integrases/*genetics
MH  - RNA/*biosynthesis/*genetics
EDAT- 2012/04/10 06:00
MHDA- 2012/12/10 06:00
CRDT- 2012/04/10 06:00
PHST- 2012/01/05 00:00 [received]
PHST- 2012/03/15 00:00 [revised]
PHST- 2012/03/20 00:00 [accepted]
PHST- 2012/04/10 06:00 [entrez]
PHST- 2012/04/10 06:00 [pubmed]
PHST- 2012/12/10 06:00 [medline]
AID - S0168-1656(12)00183-6 [pii]
AID - 10.1016/j.jbiotec.2012.03.016 [doi]
PST - ppublish
SO  - J Biotechnol. 2012 Aug 31;160(3-4):129-39. doi: 10.1016/j.jbiotec.2012.03.016. 
      Epub 2012 Mar 30.