2011
DOI: 10.1007/978-1-61779-352-3_15
|View full text |Cite
|
Sign up to set email alerts
|

Methods for Constructing Clones for Protein Expression in Mammalian Cells

Abstract: Multisite Gateway technology is a DNA cloning method based on in vitro site-specific recombination that is becoming increasingly popular because it allows quick and highly efficient assembly of multiple DNA fragments into a vector backbone. In the conventional Gateway Multisite strategy, cloning of multiple DNA fragments requires recombination of multiple entry clones with a single destination vector. The -limitation of this approach is that as the number of entry clones increases, the efficiency of the assemb… Show more

Help me understand this report

Search citation statements

Order By: Relevance

Paper Sections

Select...
2
2
1

Citation Types

0
8
0

Year Published

2011
2011
2020
2020

Publication Types

Select...
5
1

Relationship

3
3

Authors

Journals

citations
Cited by 7 publications
(8 citation statements)
references
References 12 publications
0
8
0
Order By: Relevance
“…The pcDNA-Myc-Ruc plasmid was further modified to produce a Gateway vector (pREN5-ATT), containing ATT recombination sites flanking the CCB toxic gene for accepting in-frame genes from Gateway donor clones. Gateway recombination reactions for generating the MTB antigens were then performed essentially as described [ 19 ]. Briefly, miniprep DNA was obtained from preexisting Gateway MTB donor clones grown under kanamycin selection.…”
Section: Methodsmentioning
confidence: 99%
“…The pcDNA-Myc-Ruc plasmid was further modified to produce a Gateway vector (pREN5-ATT), containing ATT recombination sites flanking the CCB toxic gene for accepting in-frame genes from Gateway donor clones. Gateway recombination reactions for generating the MTB antigens were then performed essentially as described [ 19 ]. Briefly, miniprep DNA was obtained from preexisting Gateway MTB donor clones grown under kanamycin selection.…”
Section: Methodsmentioning
confidence: 99%
“…As the original tet-inducible BclxL and miR-9/9*-124 lentiviral vector, pTight-9/ 9*-124-BclxL [24], was a second-generation lentiviral vector, we transferred the expression cassette into a third-generation self-inactivating lentiviral vector backbone. The tet-inducible all-in-one self-inactivation lentiviral vector expressing BclxL and miR-9/9*-124, CSIV-124-9/9*-BclxL-TRE-EF-KT, was constructed by a Multisite Gateway-based method as previously described [30]. The human BclxL gene or the miR-9/9*-124 was amplified by PCR by using a primer set with corresponding additional attB-signals at 5'-ends and pTight-9/9*-124-BclxL as a common template.…”
Section: Preparation and Infection Of Lentivirusesmentioning
confidence: 99%
“…The human BclxL gene or the miR-9/9*-124 was amplified by PCR by using a primer set with corresponding additional attB-signals at 5'-ends and pTight-9/9*-124-BclxL as a common template. The resultant attB-signal flanked PCR fragment, B3-hBclxL-B2 or B2r-miR-9/9*-124-B4 was cloned into pDONR-P3P2 or pDONR-P2rP4 [30], respectively, by Gateway BP cloning. The resultant entry clones, pENTR-B3-hBclxL-B2 and pENTR-B2r-miR-9/9*-124-B4, were assembled into a tet-inducible Multisite Gatewaycompatible self-inactivation lentiviral vector, CSIV-DEST-R4R3-TRE-EF-KT, by Gateway LR cloning.…”
Section: Preparation and Infection Of Lentivirusesmentioning
confidence: 99%
“…One improvement to the current strategy would be the delivery of multiple reprogramming factors within the context of a single vector. Construction of multiple functional DNA elements in tandem on a single plasmid has been performed by stepwise Gateway recombination reactions using Multisite Gateway technology (Life Technologies, Carlsbad, USA) (Cheo et al, 2004;Inoue et al, 2009;Sasaki et al, 2004;Sone and Imamoto, 2011;Sone et al, 2008).…”
Section: Introductionmentioning
confidence: 99%