Whereas bacterial artificial chromosomes (BACs) offer many advantages in studies of gene and protein function, generation of seamless, precisely mutated BACs has been difficult. Here we describe a counterselection-based recombineering method and its accompanying reagents. After identifying intramolecular recombination as the major problem in counterselection, we built a strategy to reduce these unwanted events by expressing Redβ alone at the crucial step. We enhanced this method by using phosphothioated oligonucleotides, using a sequence-altered rpsL counterselection gene and developing online software for oligonucleotide design. We illustrated this method by generating transgenic mammalian cell lines carrying small interfering RNA-resistant and point-mutated BAC transgenes. Using this approach, we generated mutated TACC3 transgenes to identify phosphorylation-specific spindle defects after knockdown of endogenous TACC3 expression. Our results highlight the complementary use of precisely mutated BAC transgenes and RNA interference in the study of cell biology at physiological expression levels and regulation.
"A 100(shown as patterned region in WT sequence) was selected for the kpsD and waaL; StrepR strains harboring the pRedET plasmid were transformed with PCR product consisting of the rpsL-neo cassette flanked by 50 bp regions homologous to sequences on either side of the chosen point of insertion; double cross-over events during homologous recombination make the mutant strains StrepS KanR due to integration of the rpsL-neo cassette; for complementation, mutant strains were transformed with 100 bp oligonucleotides bearing the same sequence as the WT across the region of insertion; complemented strains were again StrepR due to loss of the rpsL-neo cassette and restoration of the parental genotype; streptomycin resistant (StrepR), streptomycin sensitive (StrepS), kanamycin resistant (KanR). The methodology depicted in this figure is a modification of the rpsL counter-selection technique previously used for site-directed mutagenesis in bacterial artificial chromosomes –. "
[Show abstract][Hide abstract] ABSTRACT: Urinary tract infection (UTI) is one of the most common bacterial infections in humans, with uropathogenic Escherichia coli (UPEC) the leading causative organism. UPEC has a number of virulence factors that enable it to overcome host defenses within the urinary tract and establish infection. The O antigen and the capsular polysaccharide are two such factors that provide a survival advantage to UPEC. Here we describe the application of the rpsL counter selection system to construct capsule (kpsD) and O antigen (waaL) mutants and complemented derivatives of three reference UPEC strains: CFT073 (O6:K2:H1), RS218 (O18:K1:H7) and 1177 (O1:K1:H7). We observed that while the O1, O6 and O18 antigens were required for survival in human serum, the role of the capsule was less clear and linked to O antigen type. In contrast, both the K1 and K2 capsular antigens provided a survival advantage to UPEC in whole blood. In the mouse urinary tract, mutation of the O6 antigen significantly attenuated CFT073 bladder colonization. Overall, this study contrasts the role of capsule and O antigen in three common UPEC serotypes using defined mutant and complemented strains. The combined mutagenesis-complementation strategy can be applied to study other virulence factors with complex functions both in vitro and in vivo.
PLoS ONE 04/2014; 9(4):e94786. DOI:10.1371/journal.pone.0094786 · 3.23 Impact Factor
"The greatest source of unwanted mutagenesis during counterselection comes from intramolecular recombination between repeated sequences to delete the counterselectable gene. Because intramolecular recombination is inherently based on double-stranded DNA, it can be minimized during counterselection by omission of Redα, which promotes dsDNA but not ssDNA recombination, and use of single-stranded oligonucleotides (32). Previously we implemented the advantages of this strategy using rpsL and streptomycin counterselection. "
[Show abstract][Hide abstract] ABSTRACT: Recombineering, which is the use of homologous recombination for DNA engineering in Escherichia coli, usually uses antibiotic selection to identify the intended recombinant. When combined in a second step with counterselection
using a small molecule toxin, seamless products can be obtained. Here, we report the advantages of a genetic strategy using
CcdB as the counterselectable agent. Expression of CcdB is toxic to E. coli in the absence of the CcdA antidote so counterselection is initiated by the removal of CcdA expression. CcdB counterselection
is robust and does not require titrations or experiment-to-experiment optimization. Because counterselection strategies necessarily
differ according to the copy number of the target, we describe two variations. For multi-copy targets, we use two E. coli hosts so that counterselection is exerted by the transformation step that is needed to separate the recombined and unrecombined
plasmids. For single copy targets, we put the ccdA gene onto the temperature-sensitive pSC101 Red expression plasmid so that counterselection is exerted by the standard temperature
shift to remove the expression plasmid. To reduce unwanted intramolecular recombination, we also combined CcdB counterselection
with Redα omission. These options improve the use of counterselection in recombineering with BACs, plasmids and the E. coli chromosome.
Nucleic Acids Research 12/2013; 42(5). DOI:10.1093/nar/gkt1339 · 9.11 Impact Factor
"These mutations indeed abolished the interaction of GTSE1 with EB1. To test if these residues were also critical in cells for the tip-tracking activity of GTSE1, we used counterselection recombineering  to engineer the same mutations into a GTSE1-GFP BAC transgene (“GTSE1Sk”). Similar to EB1 depletion, mutation of tandem SxLP motifs in GTSE1-GFP abolished interphase GTSE1-GFP tip tracking, but not microtubule lattice association (Figure 2D, Movie S5,S6). "
[Show abstract][Hide abstract] ABSTRACT: The regulation of cell migration is a highly complex process that is often compromised when cancer cells become metastatic. The microtubule cytoskeleton is necessary for cell migration, but how microtubules and microtubule-associated proteins regulate multiple pathways promoting cell migration remains unclear. Microtubule plus-end binding proteins (+TIPs) are emerging as important players in many cellular functions, including cell migration. Here we identify a +TIP, GTSE1, that promotes cell migration. GTSE1 accumulates at growing microtubule plus ends through interaction with the EB1+TIP. The EB1-dependent +TIP activity of GTSE1 is required for cell migration, as well as for microtubule-dependent disassembly of focal adhesions. GTSE1 protein levels determine the migratory capacity of both nontransformed and breast cancer cell lines. In breast cancers, increased GTSE1 expression correlates with invasive potential, tumor stage, and time to distant metastasis, suggesting that misregulation of GTSE1 expression could be associated with increased invasive potential.
PLoS ONE 12/2012; 7(12):e51259. DOI:10.1371/journal.pone.0051259 · 3.23 Impact Factor
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