18 research outputs found
Perspectives for artificial insemination and genomics to improve global swine populations
Civilizations throughout the world continue to depend on pig meat as an important food source. Approximately 40% of the red meat consumed annually worldwide (94 million metric tons) is pig meat. Pig numbers (940 million) and consumption have increased consistent with the increasing world population (FAO 2002). In the past 50 years, research guided genetic selection and nutrition programs have had a major impact on improving carcass composition and efficiency of production in swine. The use of artificial insemination (AI) in Europe has also had a major impact on pig improvement in the past 35 years and more recently in the USA. Several scientific advances in gamete physiology and/or manipulation have been successfully utilized while others are just beginning to be applied at the production level. Semen extenders that permit the use of fresh semen for more than 5 days post-collection are largely responsible for the success of AI in pigs worldwide. Transfer of the best genetics has been enabled by use of AI with fresh semen, and to some extent, by use of AI with frozen semen over the past 25 years. Sexed semen, now a reality, has the potential for increasing the rate of genetic progress in AI programs when used in conjunction with newly developed low sperm number insemination technology. Embryo cryopreservation provides opportunities for international transport of maternal germplasm worldwide; non-surgical transfer of viable embryos in practice is nearing reality. While production of transgenic animals has been successful, the low level of efficiency in producing these animals and lack of information on multigene interactions limit the use of the technology in applied production systems. Technologies based on research in functional genomics, proteomics and cloning have significant potential, but considerable research effort will be required before they can be utilized for AI in pig production. In the past 15 years, there has been a coordinated worldwide scientific effort to develop the genetic linkage map of the pig with the goal of identifying pigs with genetic alleles that result in improved growth rate, carcass quality, and reproductive performance. Molecular genetic tests have been developed to select pigs with improved traits such as removal of the porcine stress (RYR1) syndrome, and selection for specific estrogen receptor (ESR) alleles. Less progress has been made in developing routine tests related to diseases. Major research in genomics is being pursued to improve the efficiency of selection for healthier pigs with disease resistance properties. The sequencing of the genome of the pig to identify new genes and unique regulatory elements holds great promise to provide new information that can be used in pig production. AI, in vitro embryo production and embryo transfer will be the preferred means of implementing these new technologies to enhance efficiency of pig production in the future
Development of a porcine (Sus scofa) embryo-specific microarray: array annotation and validation
Serial analysis of gene expression during elongation of the peri-implantation porcine trophectoderm (conceptus)
Characterization of the Altered Gene Expression Profile in Early Porcine Embryos Generated from Parthenogenesis and Somatic Cell Chromatin Transfer
<div><p>The <i>in vitro</i> production of early porcine embryos is of particular scientific and economic interest. In general, embryos produced from <i>in vitro</i> Assisted Reproductive Technologies (ART) manipulations, such as somatic cell chromatin transfer (CT) and parthenogenetic activation (PA), are less developmentally competent than <i>in vivo</i>âderived embryos. The mechanisms underlying the deficiencies of embryos generated from PA and CT have not been completely understood. To characterize the altered genes and gene networks in embryos generated from CT and PA, comparative transcriptomic analyses of <i>in vivo</i> (IVV) expanded blastocysts (XB), IVV hatched blastocyst (HB), PA XB, PA HB, and CT HB were performed using a custom microarray platform enriched for genes expressed during early embryonic development. Differential expressions of 1492 and 103 genes were identified in PA and CT HB, respectively, in comparison with IVV HB. The âeIF2 signallingâ, âmitochondrial dysfunctionâ, âregulation of eIF4 and p70S6K signallingâ, âprotein ubiquitinationâ, and âmTOR signallingâ pathways were down-regulated in PA HB. Dysregulation of notch signallingâassociated genes were observed in both PA and CT HB. TP53 was predicted to be activated in both PA and CT HB, as 136 and 23 regulation targets of TP53 showed significant differential expression in PA and CT HB, respectively, in comparison with IVV HB. In addition, dysregulations of several critical pluripotency, trophoblast development, and implantation-associated genes (<i>NANOG</i>, <i>GATA2</i>, <i>KRT8</i>, <i>LGMN</i>, and <i>DPP4</i>) were observed in PA HB during the blastocyst hatching process. The critical genes that were observed to be dysregulated in CT and PA embryos could be indicative of underlying developmental deficiencies of embryos produced from these technologies.</p></div
Development of a porcine (Sus scofa) embryo-specific microarray: array annotation and validation
Abstract Background The domestic pig is an important livestock species and there is strong interest in the factors that affect the development of viable embryos and offspring in this species. A limited understanding of the molecular mechanisms involved in early embryonic development has inhibited our ability to fully elucidate these factors. Next generation deep sequencing and microarray technologies are powerful tools for delineation of molecular pathways involved in the developing embryo. Results Here we present the development of a porcine-embryo-specific microarray platform created from a large expressed sequence tag (EST) analysis generated by Roche/454 next-generation sequencing of cDNAs constructed from critical stages of in vivo or in vitro porcine preimplantation embryos. Two cDNA libraries constructed from in vitro and in vivo produced preimplantation porcine embryos were normalized and sequenced using 454 Titanium pyrosequencing technology. Over one million high-quality EST sequences were obtained and used to develop the EMbryogene Porcine Version 1 (EMPV1) microarray composed of 43,795 probes. Based on an initial probe sequence annotation, the EMPV1 features 17,409 protein-coding, 473 pseudogenes, 46 retrotransposed, 2,359 non-coding RNA, 4,121 splice variants in 2,862 genes and a total of 12,324 Novel Transcript Regions (NTR). After re-annotation, the total unique genes increased from 11,961 to 16,281 and 1.9% of them belonged to a large olfactory receptor (OR) gene family. Quality control on the EMPV1 was performed and revealed an even distribution of ten clusters of spiked-in control spots and array to array (dye-swap) correlation was 0.97. Conclusions Using next-generation deep sequencing we have produced a large EST dataset to allow for the selection of probe sequences for the development of the EMPV1 microarray platform. The quality of this embryo-specific array was confirmed with a high-level of reproducibility using current Agilent microarray technology. With more than an estimated 20,000 unique genes represented on the EMPV1, this platform will provide the foundation for future research into the in vivo and in vitro factors that affect the viability of porcine embryos, as well as the effects of these factors on the live offspring that result from these embryos.</p
Porcine induced pluripotent stem cells produce chimeric offspring
Ethical and moral issues rule out the use of human induced pluripotent stem cells (iPSCs) in chimera studies that would determine the full extent of their reprogrammed state, instead relying on less rigorous assays such as teratoma formation and differentiated cell types. To date, only mouse iPSC lines are known to be truly pluripotent. However, initial mouse iPSC lines failed to form chimeric offspring, but did generate teratomas and differentiated embryoid bodies, and thus these specific iPSC lines were not completely reprogrammed or truly pluripotent. Therefore, there is a need to address whether the reprogramming factors and process used eventually to generate chimeric mice are universal and sufficient to generate reprogrammed iPSC that contribute to chimeric offspring in additional species. Here we show that porcine mesenchymal stem cells transduced with 6 human reprogramming factors (POU5F1, SOX2, NANOG, KLF4, LIN28, and C-MYC) injected into preimplantation-stage embryos contributed to multiple tissue types spanning all 3 germ layers in 8 of 10 fetuses. The chimerism rate was high, 85.3% or 29 of 34 live offspring were chimeras based on skin and tail biopsies harvested from 2-to 5-day-old pigs. The creation of pluripotent porcine iPSCs capable of generating chimeric offspring introduces numerous opportunities to study the facets significantly affecting cell therapies, genetic engineering, and other aspects of stem cell and developmental biology
Primer sequences used in Real-time PCR verification.
<p>Primer sequences used in Real-time PCR verification.</p
QPCR verification result.
<p>QPCR verification result of 14 selected genes. The mRNA expression levels of these genes were normalized with the external control gene (Xeno), and were calculated with 2<sup>âÎÎCt</sup> relative quantification. Bar charts showing the relative expression levels of PSEN2, ANXA8, HES1, JAG1, HEY2, NCSTN, KRT18, KRT8, GATA2, NANOG, SLC36A2, KCTD3, DPP4, and LGMN genes in IVV XB, IVV HB, PA XB, PA HB, and CT HB (KCTD3, SLC36A2, and LGMN genes were not tested in CT HB). The relative expression levels of in each sample were standardized with their expression Error bars shows the standard error (*: P < 0.05). Dashed lines indicate 1.0 expression level. ND: not detected. NT: not tested.</p
Altered biological function categories in PA and CT-derived HB.
<p>Bar chart shows the significantly altered biological function categories in IPA biological function (bio-function) analysis. Major Y axis on the left shows the number of differentially expressed genes that involved in the biological function category. Secondary Y axis on the right shows the significance (-log (B-H P-value)) of the altered biological function category. The orange line shows the significance threshold of cut off of -log (B-H P-valueâ=â0.05).</p