PMID- 23281894
OWN - NLM
STAT- MEDLINE
DCOM- 20130808
LR  - 20220129
IS  - 1472-6750 (Electronic)
IS  - 1472-6750 (Linking)
VI  - 13
DP  - 2013 Jan 3
TI  - A simplified counter-selection recombineering protocol for creating fluorescent 
      protein reporter constructs directly from C. elegans fosmid genomic clones.
PG  - 1
LID - 10.1186/1472-6750-13-1 [doi]
AB  - BACKGROUND: Recombineering is a genetic engineering tool that enables facile 
      modification of large episomal clones, e.g. BACs, fosmids. We have previously 
      adapted this technology to generate, directly from fosmid-based genomic clones, 
      fusion gene reporter constructs designed to investigate gene expression patterns 
      in C. elegans. In our adaptation a rpsL-tet(A) positive/negative-selection 
      cassette (RT-cassette) is first inserted and then, under negative selection, 
      seamlessly replaced with the desired sequence. We report here on the generation 
      and application of a resource comprising two sets of constructs designed to 
      facilitate this particular recombineering approach. RESULTS: Two complementary 
      sets of constructs were generated. The first contains different fluorescent 
      protein reporter coding sequences and derivatives while the second set of 
      constructs, based in the copy-number inducible vector pCC1Fos, provide a resource 
      designed to simplify RT-cassette-based recombineering. These latter constructs 
      are used in pairs the first member of which provides a template for 
      PCR-amplification of an RT-cassette while the second provides, as an excised 
      restriction fragment, the desired fluorescent protein reporter sequence. As the 
      RT-cassette is flanked by approximately 200 bp from the ends of the reporter 
      sequence the subsequent negative selection replacement step is highly efficient. 
      Furthermore, use of a restriction fragment minimizes artefacts negating the need 
      for final clone sequencing. Utilizing this resource we generated single-, double- 
      and triple-tagged fosmid-based reporters to investigate expression patterns of 
      three C. elegans genes located on a single genomic clone. CONCLUSIONS: We 
      describe the generation and application of a resource designed to facilitate 
      counter-selection recombineering of fosmid-based C. elegans genomic clones. By 
      choosing the appropriate pair of 'insertion' and 'replacement' constructs 
      recombineered products, devoid of artefacts, are generated at high efficiency. 
      Gene expression patterns for three genes located on the same genomic clone were 
      investigated via a set of fosmid-based reporter constructs generated with the 
      modified protocol.
FAU - Hirani, Nisha
AU  - Hirani N
AD  - Institute of Pharmaceutical Science, King's College London, Franklin-Wilkins 
      Building, 150 Stamford Street, London, SE1 9NH, UK.
FAU - Westenberg, Marcel
AU  - Westenberg M
FAU - Gami, Minaxi S
AU  - Gami MS
FAU - Davis, Paul
AU  - Davis P
FAU - Hope, Ian A
AU  - Hope IA
FAU - Dolphin, Colin T
AU  - Dolphin CT
LA  - eng
GR  - G0701197/MRC_/Medical Research Council/United Kingdom
GR  - BB/E008038/1/BB_/Biotechnology and Biological Sciences Research Council/United 
      Kingdom
GR  - WT082603/WT_/Wellcome Trust/United Kingdom
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
DEP - 20130103
PL  - England
TA  - BMC Biotechnol
JT  - BMC biotechnology
JID - 101088663
RN  - 0 (Luminescent Proteins)
SB  - IM
MH  - Animals
MH  - Caenorhabditis elegans/*genetics
MH  - Gene Expression
MH  - Genes, Reporter
MH  - Genetic Engineering/*methods
MH  - Genetic Vectors/*metabolism
MH  - *Genome
MH  - Luminescent Proteins/genetics/*metabolism
PMC - PMC3561212
EDAT- 2013/01/04 06:00
MHDA- 2013/08/09 06:00
PMCR- 2013/01/03
CRDT- 2013/01/04 06:00
PHST- 2012/06/25 00:00 [received]
PHST- 2012/12/07 00:00 [accepted]
PHST- 2013/01/04 06:00 [entrez]
PHST- 2013/01/04 06:00 [pubmed]
PHST- 2013/08/09 06:00 [medline]
PHST- 2013/01/03 00:00 [pmc-release]
AID - 1472-6750-13-1 [pii]
AID - 10.1186/1472-6750-13-1 [doi]
PST - epublish
SO  - BMC Biotechnol. 2013 Jan 3;13:1. doi: 10.1186/1472-6750-13-1.