Creating Porcine Biomedical Models Through Recombineering

Recent advances in genomics provide genetic information from humans and other mammals (mouse, rat, dog and primates) traditionally used as models as well as new candidates (pigs and cattle). In addition, linked enabling technologies, such as transgenesis and animal cloning, provide innovative ways to design and perform experiments to dissect complex biological systems. Exploitation of genomic information overcomes the traditional need to choose naturally occurring models. Thus, investigators can utilize emerging genomic knowledge and tools to create relevant animal models. This approach is referred to as reverse genetics. In contrast to ‘forward genetics’, in which gene(s) responsible for a particular phenotype are identified by positional cloning (phenotype to genotype), the ‘reverse genetics’ approach determines the function of a gene and predicts the phenotype of a cell, tissue, or organism (genotype to phenotype). The convergence of classical and reverse genetics, along with genomics, provides a working definition of a ‘genetic model’ organism (3). The recent construction of phenotypic maps defining quantitative trait loci (QTL) in various domesticated species provides insights into how allelic variations contribute to phenotypic diversity. Targeted chromosomal regions are characterized by the construction of bacterial artificial chromosome (BAC) contigs to isolate and characterize genes contributing towards phenotypic variation. Recombineering provides a powerful methodology to harvest genetic information responsible for phenotype. Linking recombineering with gene-targeted homologous recombination, coupled with nuclear transfer (NT) technology can provide ‘clones’ of genetically modified animals

A high utility integrated map of the pig genome

International audienc

Piggy-BACing the human genome I: constructing a porcine BAC physical map through comparative genomics

Availability of the human genome sequence and high similarity between humans and pigs at the molecular level provides an opportunity to use a comparative mapping approach to piggy-BAC the human genome. In order to advance the pig genome sequencing initiative, sequence similarity between large-scale porcine BAC-end sequences (BESs) and human genome sequence was used to construct a comparatively-anchored porcine physical map that is a first step towards sequencing the pig genome. A total of 50,300 porcine BAC clones were end-sequenced, yielding 76,906 BESs after trimming with an average read length of 538 bp. To anchor the porcine BACs on the human genome, these BESs were subjected to BLAST analysis using the human draft sequence, revealing 31.5% significant hits (E < e⁻⁵). Both genic and non-genic regions of homology contributed to the alignments between the human and porcine genomes. Porcine BESs with unique homology matches within the human genome provided a source of markers spaced approximately 70 to 300 kb along each human chromosome. In order to evaluate the utility of piggy-BACing human genome sequences, and confirm predictions of orthology, 193 evenly spaced BESs with similarity to HSA3 and HSA21 were selected and then utilized for developing a high-resolution (1.22 Mb) comparative radiation hybrid map of SSC13 that represents a fusion of HSA3 and HSA21. Resulting RH mapping of SSC13 covers 99% and 97% of HSA3 and HSA21, respectively. Seven evolutionary conserved blocks were identified including six on HSA3 and a single syntenic block corresponding to HSA21. The strategy of piggy-BACing the human genome described in this study demonstrates that through a directed, targeted comparative genomics approach construction of a high-resolution anchored physical map of the pig genome can be achieved. This map supports the selection of BACs to construct a minimal tiling path for genome sequencing and targeted gap filling. Moreover, this approach is highly relevant to other genome sequencing projects.Margarita B. Rogatcheva; Kefei Chen; Denis M. Larkin; Stacey N. Meyers; Brandy M. Marron; Weisong He; Lawrence B. Schook; Jonathan E. Beeve

Piggy-BACing the human genome: II. A high-resolution, physically anchored, comparative map of the porcine autosomes

Using the INRA-Minnesota porcine radiation hybrid panel, we have constructed a human-pig comparative map composed of 2274 loci, including 206 ESTs and 2068 BAC-end sequences, assigned to 34 linkage groups. The average spacing between comparative anchor loci is 1.15 Mb based on human genome sequence coordinates. A total of 51 conserved synteny groups that include 173 conserved segments were identified. This radiation hybrid map has the highest resolution of any porcine map to date and its integration with the porcine linkage map (reported here) will greatly facilitate the positional cloning of genes influencing complex traits of both agricultural and biomedical interest. Additionally, this map will provide a framework for anchoring contigs generated through BAC fingerprinting efforts and assist in the selection of a BAC minimal tiling path and assembly of the first sequence-ready map of the porcine genome

Dynamics of Mammalian Chromosome Evolution Inferred from Multispecies Comparative Maps

The genome organizations of eight phylogenetically distinct species from five mammalian orders were compared in order to address fundamental questions relating to mammalian chromosomal evolution. Rates of chromosome evolution within mammalian orders were found to increase since the Cretaceous-Tertiary boundary. Nearly 20% of chromosome breakpoint regions were reused during mammalian evolution; these reuse sites are also enriched for centromeres. Analysis of gene content in and around evolutionary breakpoint regions revealed increased gene density relative to the genome-wide average. We found that segmental duplications populate the majority of primate-specific breakpoints and often flank inverted chromosome segments, implicating their role in chromosomal rearrangement