Annotation of the Domestic Pig Genome by Quantitative Proteogenomics.

The pig is one of the earliest domesticated animals in the history of human civilization and represents one of the most important livestock animals. The recent sequencing of the Sus scrofa genome was a major step toward the comprehensive understanding of porcine biology, evolution, and its utility as a promising large animal model for biomedical and xenotransplantation research. However, the functional and structural annotation of the Sus scrofa genome is far from complete. Here, we present mass spectrometry-based quantitative proteomics data of nine juvenile organs and six embryonic stages between 18 and 39 days after gestation. We found that the data provide evidence for and improve the annotation of 8176 protein-coding genes including 588 novel and 321 refined gene models. The analysis of tissue-specific proteins and the temporal expression profiles of embryonic proteins provides an initial functional characterization of expressed protein interaction networks and modules including as yet uncharacterized proteins. Comparative transcript and protein expression analysis to human organs reveal a moderate conservation of protein translation across species. We anticipate that this resource will facilitate basic and applied research on Sus scrofa as well as its porcine relatives.

Non-invasive assessment of porcine oocyte quality by supravital staining of cumulus-oocyte complexes with lissamine green B.

We evaluated the usefulness of lissamine green B (LB) staining of cumulus-oocyte complexes (COC) as a non-invasive method of predicting maturational and developmental competence of slaughterhouse-derived porcine oocytes cultured in vitro. Cumulus cells of freshly aspirated COCs were evaluated either morphologically on the basis of thickness of cumulus cell layers, or stained with LB, which penetrates only non-viable cells. The extent of cumulus cell staining was taken as an inverse indicator of membrane integrity. The two methods of COC grading were then examined as predictors of nuclear maturation and development after parthenogenetic activation. In both cases LB staining proved a more reliable indicator than morphological assessment (P < 0.05). The relationship between LB staining and cumulus cell apoptosis was also examined. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay for DNA fragmentation revealed that oocytes within COCs graded as low quality by either LB staining or visual morphology showed significantly greater DNA fragmentation (P < 0.05) than higher grades, and that LB and visual grading were of similar predictive value. Expression of the stress response gene TP53 showed significantly higher expression in COCs graded as low quality by LB staining. However expression of the apoptosis-associated genes BAK and CASP3 was not significantly different between high or low grade COCs, suggesting that mRNA expression of BAK and CASP3 is not a reliable method of detecting apoptosis in porcine COCs. Evaluation of cumulus cell membrane integrity by lissamine green B staining thus provides a useful new tool to gain information about the maturational and developmental competence of porcine oocytes.

A porcine model of familial adenomatous polyposis.

We created gene-targeted pigs with mutations in the adenomatous polyposis coli (APC) gene (APC) that are orthologous to those responsible for human familial adenomatous polyposis (FAP). One-year-old pigs with the APC(1311) mutation (orthologous to human APC(1309)) have aberrant crypt foci and low- and high-grade dysplastic adenomas in the large intestine, similar to the precancerous lesions that develop in patients with FAP. Dysplastic adenomas accumulate ß-catenin and lose heterozygosity of APC. This large-animal, genetic model of FAP will be useful in the development of diagnostics and therapeutics for colorectal cancer. DNA sequence data: NCBI accession number GU951771.

Efficient immunoglobulin gene disruption and targeted replacement in rabbit using zinc finger nucleases.

Rabbits are widely used in biomedical research, yet techniques for their precise genetic modification are lacking. We demonstrate that zinc finger nucleases (ZFNs) introduced into fertilized oocytes can inactivate a chosen gene by mutagenesis and also mediate precise homologous recombination with a DNA gene-targeting vector to achieve the first gene knockout and targeted sequence replacement in rabbits. Two ZFN pairs were designed that target the rabbit immunoglobulin M (IgM) locus within exons 1 and 2. ZFN mRNAs were microinjected into pronuclear stage fertilized oocytes. Founder animals carrying distinct mutated IgM alleles were identified and bred to produce offspring. Functional knockout of the immunoglobulin heavy chain locus was confirmed by serum IgM and IgG deficiency and lack of IgM(+) and IgG(+) B lymphocytes. We then tested whether ZFN expression would enable efficient targeted sequence replacement in rabbit oocytes. ZFN mRNA was co-injected with a linear DNA vector designed to replace exon 1 of the IgM locus with ∼1.9 kb of novel sequence. Double strand break induced targeted replacement occurred in up to 17% of embryos and in 18% of fetuses analyzed. Two major goals have been achieved. First, inactivation of the endogenous IgM locus, which is an essential step for the production of therapeutic human polyclonal antibodies in the rabbit. Second, establishing efficient targeted gene manipulation and homologous recombination in a refractory animal species. ZFN mediated genetic engineering in the rabbit and other mammals opens new avenues of experimentation in immunology and many other research fields.

Molecular phylogeny of Megaloceros giganteus--the giant deer or just a giant red deer?

Two fragments of mitochondrial DNA (mtDNA) of the cytochrome b gene (137 bp and 167 bp) were successfully isolated and sequenced from antlers and bones of five specimens of the Giant Deer (Megaloceros giganteus) to examine the phylogenetic position of Megaloceros giganteus within the family Cervidae. This is the first report on ancient DNA (aDNA) sequences from Megaloceros giganteus. A phylogenetic analysis based on parameter-rich models describes the evolutionary relationships between five individuals of fossil Megaloceros giganteus and 37 individuals of 11 extant species of the family Cervidae. The results support a "Cervus-Megaloceros" clade. The phylogenetic positions of sympatric Megaloceros and Cervus elaphus specimens in particular indicate either that the Megaloceros mtDNA gene pool did not evolve for a substantial time period as an entity distinct from Cervus elaphus until its extinction, or that Megaloceros contributed mtDNA to Cervus elaphus or vice versa. The results of this study allow the conclusion that the European Megaloceros giganteus is more related to its modern regional counterparts of the species of Cervus elaphus than recent claims have suggested.

QTLs for pre- and postweaning body weight and body composition in selected mice.

In an intercross between the high-body-weight-selected mouse line NMRI8 and the inbred line DBA/2, we analyzed genetic effects on growth during the suckling period and after weaning during the juvenile phase of development. QTL mapping results indicated that a switch of gene activation might occur at the age of three weeks when animals are weaned. We found QTLs for body weight with major effects at the age of two and three weeks when animals are fed by their mothers, and QTLs with highest effects after weaning when animals have to live on their own under ad libitum access to food. Specific epistatic effects on body weight at two and three weeks and epistatic interaction influencing growth after weaning support this finding. QTL effects explained the greatest variance during puberty when animals grow fastest and become fertile. In the present study, all except one QTL effect for early body weight had dominance variance components. These might result from direct single-locus-dominant allelic expression, but also from the identified epistatic interaction between different QTLs that we have found for body weight at all ages. Beside body weight, body composition traits (muscle weight, reproductive fat weight, weight of inner organs) were analyzed. Sex-dimorphic QTLs were found for body weight and fat deposition. The identified early-growth QTLs could be the target of epigenetic modifications which might influence body weight at later ages.

Mitochondrial DNA phylogeography of red deer (Cervus elaphus).

In order to understand the origin, phylogeny, and phylogeography of the species Cervus elaphus, we examined the DNA sequence variation of the mitochondrial cytochrome b gene of 51 populations of deer from the entire distribution area of Cervinae with an emphasis on Europe and Asia. Several methods, including maximum parsimony, maximum likelihood, and nested clade analysis, revealed that red deer originated from the area between Kyrgyzstan and Northern India. We found two distinct groups of red deer: a western group consisting of four subgroups and an eastern group consisting of three subgroups. Our mtDNA data do not support the traditional classification of red deer as only one species nor its division into numerous subspecies. The discrepancies between the geographical pattern of differentiation based on mtDNA cytochrome b and the existing specific and subspecific taxonomy based on morphology are discussed.

Differentiation of Castor fiber and Castor Canadensis by noninvasive molecular methods.

The aim of the study was to develop a simple and reliable method for differentiation of the two phenotypic, very similar Eurasian and North American beavers. Hair bulbs were plucked as tissue samples from the fur of living animals. The mitochondrial cytochrome b locus was amplified by polymerase chain reaction and sequenced. The fragments of the two species differed at 44/41 nucleotide sites. RsaI recognised two mutations, resulting in a restriction fragment length polymorphism that seems to be species specific, as could be revealed by the banding pattern. Zoo Biol 19:511-515, 2000. Copyright 2000 Wiley-Liss, Inc.