Targeted isolation of cloned genomic regions by recombineering for haplotype phasing and isogenic targeting
ABSTRACT Studying genetic variations in the human genome is important for understanding phenotypes and complex traits, including rare personal variations and their associations with disease. The interpretation of polymorphisms requires reliable methods to isolate natural genetic variations, including combinations of variations, in a format suitable for downstream analysis. Here, we describe a strategy for targeted isolation of large regions (∼35 kb) from human genomes that is also applicable to any genome of interest. The method relies on recombineering to fish out target fosmid clones from pools and thereby circumvents the laborious need to plate and screen thousands of individual clones. To optimize the method, a new highly recombineering-efficient bacterial host, including inducible TrfA for fosmid copy number amplification, was developed. Various regions were isolated from human embryonic stem cell lines and a personal genome, including highly repetitive and duplicated ones. The maternal and paternal alleles at the MECP2/IRAK 1 loci were distinguished based on identification of novel allele-specific single-nucleotide polymorphisms in regulatory regions. Additionally, we applied further recombineering to construct isogenic targeting vectors for patient-specific applications. These methods will facilitate work to understand the linkage between personal variations and disease propensity, as well as possibilities for personal genome surgery.
SourceAvailable from: Andrei I. Pozniakovsky
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ABSTRACT: Efficient gene targeting in embryonic stem cells requires that modifying DNA sequences are identical to those in the targeted chromosomal locus. Yet, there is a paucity of isogenic genomic clones for human cell lines and PCR amplification cannot be used in many mutation-sensitive applications. Here, we describe a novel method for the direct cloning of genomic DNA into a targeting vector, pRTVIR, using oligonucleotide-directed homologous recombination in yeast. We demonstrate the applicability of the method by constructing functional targeting vectors for mammalian genes Uhrf1 and Gfap. Whereas the isogenic targeting of the gene Uhrf1 showed a substantial increase in targeting efficiency compared to non-isogenic DNA in mouse E14 cells, E14-derived DNA performed better than the isogenic DNA in JM8 cells for both Uhrf1 and Gfap. Analysis of 70 C57BL/6-derived targeting vectors electroporated in JM8 and E14 cell lines in parallel showed a clear dependence on isogenicity for targeting, but for three genes isogenic DNA was found to be inhibitory. In summary, this study provides a straightforward methodological approach for the direct generation of isogenic gene targeting vectors.PLoS ONE 09/2013; 8(9):e74207. DOI:10.1371/journal.pone.0074207 · 3.53 Impact Factor
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ABSTRACT: Next-generation sequencing technologies have led to rapid progress in the fields of human and nonhuman primate (NHP) genomics. The less expensive and more efficient technologies have enabled the sequencing of human genomes from multiple populations and the sequencing of many NHP species. NHP genomes have been sequenced for two main reasons: (1) their importance as animal models in biomedical research and (2) their phylogenetic relationship to humans and use in derivative evolutionary studies. NHPs are valuable animal models for a variety of diseases, most notably for human immunodeficiency virus/acquired immunodeficiency syndrome research, and for vaccine development. Knowledge about the variation in primate immune response loci can provide essential insights into relevant immune function. However, perhaps ironically considering their central role in infectious disease, the accumulation of sequence detail from genomic regions harboring immune response loci, such as the major histocompatibility complex and killer immunoglobulin-like receptors, has been slow. This deficiency is, at least in part, due to the highly repetitive and polymorphic nature of these regions and is being addressed by the application of special approaches to targeted sequencing of the immune response genomic regions. We discuss one such targeting approach that has successfully yielded complete phased genomic sequences from complex genomic regions and is now being used to resequence macaque and other primate major histocompatibility complex regions. The essential detail contained within the genomics of the NHP immune response is now being assembled, and the realization of precise comparisons between NHP and human immune genomics is close at hand, further enhancing the NHP animal model in the search for effective treatments for human disease.ILAR journal / National Research Council, Institute of Laboratory Animal Resources 01/2013; 54(2):181-95. DOI:10.1093/ilar/ilt043 · 1.05 Impact Factor