DNA gap repair in Escherichia coli for multiplex site-directed mutagenesis.

Site-directed mutagenesis allows the generation of novel DNA sequences that can be used for a variety of important applications such as the functional analysis of genetic variants. To overcome the limitations of existing site-directed mutagenesis approaches, we explored in vivo DNA gap repair. We found that site-specific mutations in plasmid DNA can be generated in Escherichia coli using mutant single-stranded oligonucleotides to target PCR-derived linear double-stranded plasmid DNA.

Live-cell PCR and one-step purification streamline DNA engineering.

In vivo DNA engineering such as recombineering (recombination-mediated genetic engineering) and DNA gap repair typically involve growing Escherichia coli (E coli) containing plasmids, followed by plasmid DNA extraction and purification prior to downstream PCR-mediated DNA modifications and DNA sequencing. We previously demonstrated that crude cell lysates could be used for some limited downstream DNA applications. Here, we show how live E coli cell PCR and one-step LiCl-isopropanol purification can streamline DNA engineering.

Direct Isolation of Seamless Mutant Bacterial Artificial Chromosomes.

Seamless (i.e., without unwanted DNA sequences) mutant bacterial artificial chromosomes (BACs) generated via recombination-mediated genetic engineering (recombineering) are better suited to study gene function compared to complementary DNA (cDNA) because they contain only the specific mutation and provide all the regulatory sequences required for in vivo gene expression.

Nodal signaling from the visceral endoderm is required to maintain Nodal gene expression in the epiblast and drive DVE/AVE migration.

In the early mouse embryo, a specialized population of extraembryonic visceral endoderm (VE) cells called the distal VE (DVE) arises at the tip of the egg cylinder stage embryo and then asymmetrically migrates to the prospective anterior, recruiting additional distal cells. Upon migration these cells, called the anterior VE (AVE), establish the anterior posterior (AP) axis by restricting gastrulation-inducing signals to the opposite pole. The Nodal-signaling pathway has been shown to have a critical role in the generation and migration of the DVE/AVE.

Isolation of rare recombinants without using selectable markers for one-step seamless BAC mutagenesis.

Current methods to isolate rare (1:10,000-1:100,000) bacterial artificial chromosome (BAC) recombinants require selectable markers. For seamless BAC mutagenesis, selectable markers need to be removed after isolation of recombinants through counterselection. Here we illustrate founder principle-driven enrichment (FPE), a simple method to rapidly isolate rare recombinants without using selectable markers, allowing one-step seamless BAC mutagenesis.