High-efficiency counterselection recombineering for site-directed mutagenesis in bacterial artificial chromosomes
ABSTRACT 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.
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ABSTRACT: Selection-based recombineering is a flexible and proven technology to precisely modify bacterial genomes at single base resolution. It consists of two steps of homologous recombination followed by selection/counter-selection. However, the shortage of efficient counter-selectable markers limits the throughput of this method. Additionally, the emergence of 'selection escapees' can affect recombinant pools generated through this method, and they must be manually removed at each step of selection-based recombineering. Here, we report a series of efforts to improve the throughput and robustness of selection-based recombineering and to achieve seamless and automatable genome engineering. Using the nucleoside kinase activity of herpes simplex virus thymidine kinase (hsvTK) on the non-natural nucleoside dP, a highly efficient, rapid, and liquid-based counter-selection system was established. By duplicating hsvtk gene, combined with careful control of the population size for the subsequent round, we effectively eliminated selection escapes, enabling seamless and multiple insertions/replacement of gene-size fragments in the chromosome. Four rounds of recombineering could thus be completed in 10 days, requiring only liquid handling and without any need for colony isolation or genotype confirmation. The simplicity and robustness of our method make it broadly accessible for multi-locus chromosomal modifications.PLoS ONE 03/2015; 10(3):e0119818. DOI:10.1371/journal.pone.0119818 · 3.53 Impact Factor
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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 · 3.53 Impact Factor
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ABSTRACT: Recombineering has been an essential tool for genetic engineering in microbes for many years and has enabled faster, more efficient engineering than previous techniques. There have been numerous studies that focus on improving recombineering efficiency, which can be divided into three main areas: (i) optimizing the oligo used for recombineering to enhance replication fork annealing and limit proofreading; (ii) mechanisms to modify the replisome itself, enabling an increased rate of annealing; and (iii) multiplexing recombineering targets and automation. These efforts have increased the efficiency of recombineering several hundred-fold. One area that has received far less attention is the problem of multiple chromosomes, which effectively decrease efficiency on a chromosomal basis, resulting in more sectored colonies, which require longer outgrowth to obtain clonal populations. Herein, we describe the problem of multiple chromosomes, discuss calculations predicting how many generations are needed to obtain a pure colony, and how changes in experimental procedure or genetic background can minimize the effect of multiple chromosomes.Biotechnology Journal 05/2013; 8(5). DOI:10.1002/biot.201200237 · 3.71 Impact Factor