Jorge A. Piedrahita

Mice lacking the folic acid-binding protein Folbp1 are defective in early embryonic development.

Periconceptional folic acid supplementation reduces the occurrence of several human congenital malformations, including craniofacial, heart and neural tube defects. Although the underlying mechanism is unknown, there may be a maternal-to-fetal folate-transport defect or an inherent fetal biochemical disorder that is neutralized by supplementation. Previous experiments have identified a folate-binding protein (Folbp1) that functions as a membrane receptor to mediate the high-affinity internalization and delivery of folate to the cytoplasm of the cell. In vitro, this receptor facilitates the accumulation of cellular folate a thousand-fold relative to the media, suggesting that it may be essential in cytoplasmic folate delivery in vivo. The importance of an adequate intracellular folate pool for normal embryogenesis has long been recognized in humans and experimental animals. To determine whether Folbp1 is involved in maternal-to-fetal folate transport, we inactivated Folbp1 in mice. We also produced mice lacking Folbp2, another member of the folate receptor family that is GPI anchored but binds folate poorly. Folbp2–/– embryos developed normally, but Folbp1–/– embryos had severe morphogenetic abnormalities and died in utero by embryonic day (E) 10. Supplementing pregnant Folbp1+/– dams with folinic acid reversed this phenotype in nullizygous pups. Our results suggest that Folbp1 has a critical role in folate homeostasis during development, and that functional defects in the human homologue (FOLR1) of Folbp1 may contribute to similar defects in humans.

A Highly Efficient Method for Porcine Cloning by Nuclear Transfer Using In Vitro-Matured Oocytes

To date, the efficiency of pig cloning by nuclear transfer of somatic cell nuclei has been extremely low, with less than 1% of transferred embryos surviving to term. Even the utilization of complex procedures such as two rounds of nuclear transfer has not resulted in greater overall efficiencies. As a result, the applicability of the technology for the generation of transgenic and cloned animals has not moved forward rapidly. We report here a simple nuclear transfer protocol, utilizing commercially available in vitro-matured oocytes, that results in greater than 5% overall cloning efficiency. Of five recipients receiving nuclear transfer embryos produced with a fetal fibroblast cell line as nuclear donor, all five established pregnancies by day 28 (100%), and 4/5 (80%) went to term. Efficiencies for each transfer were 7% (9 piglets/128 doublets transferred), 5% (5/100), 12% (7/59), and 6.6% (7/106). The overall efficiency in all recipients was 5.5% and in pregnant recipients 7.7%, with a total of 28 cloned piglets produced. With the average fusion rate being 58%, the percentage of fused doublets producing a live piglet approached 12%. The method described here can be undertaken by a single micromanipulator at a reasonable cost, and should facilitate the broad utilization of porcine cloning technology in transgenic and nontransgenic applications.

Epigenetic and Genomic Imprinting Analysis in Nuclear Transfer Derived Bos gaurus/Bos taurus Hybrid Fetuses

Abstract Somatic cell nuclear transfer (NT) in cattle is an inefficient process, whereby the production of calves is hindered by low pregnancy rates as well as fetal and placental abnormalities. Interspecies models have been previously used to facilitate the identification of single nucleotide polymorphisms (SNPs) within coding regions of genes to discriminate between parental alleles in the offspring. Here we report the use of a bovine interspecies model (Bos gaurus × Bos taurus) for the assessment and characterization of epigenetic modifications and genomic imprinting in Day 40-old female NT-derived fetuses and placenta. Analysis of NT and control pregnancies indicated disruption of genomic imprinting at the X inactivation-specific transcript (XIST) locus in the chorion, but not the fetus of clones, whereas proper allelic expression of the insulin-like growth factor II (IGF2) and gene trap locus 2 (GTL2) loci was maintained in both the fetus and placenta. Analysis of the XIST differentially methylated region (DMR) in clones indicated normal patterns of methylation; however, bisulfite sequencing of the satellite I repeat element and epidermal cytokeratin promoter indicated hypermethylation in the chorion of clones when compared with controls. No differences were detected in methylation levels in the fetus proper. These results indicate that the nuclear transfer process affects gene expression patterns in the trophectoderm- and inner cell mass-derived tissues to different extents.

Neural Tube and Craniofacial Defects With Special Emphasis On Folate Pathway Genes

Neural tube and orofacial defects are common congenital malformations in humans. While etiologically heterogeneous, they are for the most part multifactorial in their pathogenesis, having both genetic and environmental components in their development. In recent years, there has been a great deal of epidemiologic evidence demonstrating that women who received multivitamins containing folic acid periconceptionally had significantly reduced occurrence and recurrence risks for producing infants with such malformations. This risk reduction is not observed in all populations, further suggestive of a genetic regulation of this phenomenon. Unfortunately, the mechanisms underlying the beneficial effects of folic acid are not well-understood. In this article, we review the relevant epidemiologic data on both neural tube defects and orofacial malformations, the fundamental embryological processes involved in closing the neural tube, and the development of the craniofacies, and propose a working hypothesis for susceptibility to these malformations. This hypothesis is based on the interworkings of cellular folate transport, focusing on the key elements involved in potocytosis. We propose that infants with mutations in the folate receptor alpha gene might be at increased risk for congenital anomalies due to a reduced binding affinity for 5-methyltetrahydrofolate, the physiologic form of folic acid. Various experimental approaches to test the working hypothesis are considered.

Hierarchical Phenotypic and Epigenetic Variation in Cloned Swine

Abstract Cloning by somatic cell nuclear transfer can result in the birth of animals with phenotypic and gene expression abnormalities. We compared adult cloned pigs and adult pigs from naturally bred control females using a series of physiological and genetic parameters, including detailed methylation profiles of selected genomic regions. Phenotypic and genetic analyses indicated that there are two classes of traits, one in which the cloned pigs have less variation than controls and another characterized by variation that is equally high in cloned and control pigs. Although cloning creates animals within the normal phenotypic range, it increases the variability associated with some traits. This finding is contrary to the expectation that cloning can be used to reduce the size of groups involved in animal experimentation and to reproduce an animal, including a pet, with a homogenous set of desired traits.

Cloning to reproduce desired genotypes

Cloned sheep, cattle, goats, pigs and mice have now been produced using somatic cells for nuclear transplantation. Animal cloning is still very inefficient with on average less than 10% of the cloned embryos transferred resulting in a live offspring. However successful cloning of a variety of different species and by a number of different laboratory groups has generated tremendous interest in reproducing desired genotypes. Some of these specific genotypes represent animal cell lines that have been genetically modified. In other cases there is a significant demand for cloning animals characterized by their inherent genetic value, for example prize livestock, household pets and rare or endangered species. A number of different variables may influence the ability to reproduce a specific genotype by cloning. These include species, source of recipient ova, cell type of nuclei donor, treatment of donor cells prior to nuclear transfer, and the techniques employed for nuclear transfer. At present, there is no solid evidence that suggests cloning will be limited to only a few specific animals, and in fact, most data collected to date suggests cloning will be applicable to a wide variety of different animals. The ability to reproduce any desired genotype by cloning will ultimately depend on the amount of time and resources invested in research.

Characterization of Conserved and Nonconserved Imprinted Genes in Swine

To increase our understanding of imprinted genes in swine, we carried out a comprehensive analysis of this gene family using two complementary approaches: expression and phenotypic profiling of parthenogenetic fetuses, and analysis of imprinting by pyrosequencing. The parthenote placenta and fetus were smaller than those of controls but had no obvious morphological differences at Day 28 of gestation. By Day 30, however, the parthenote placentas had decreased chorioallantoic folding, decreased chorionic ruggae, and reduction of fetal-maternal interface surface in comparison with stage-matched control fetuses. Using Affymetrix Porcine GeneChip microarrays and/or semiquantitative PCR, brain, fibroblast, liver, and placenta of Day 30 fetuses were profiled, and 25 imprinted genes were identified as differentially expressed in at least one of the four tissue types: AMPD3, CDKN1C, COPG2, DHCR7, DIRAS3, IGF2 (isoform specific), IGF2AS, IGF2R, MEG3, MEST, NAP1L5, NDN, NNAT, OSBPL1A, PEG3, APEG3, PEG10, PLAGL1, PON2, PPP1R9A, SGCE, SLC38A4, SNORD107, SNRPN, and TFPI2. For DIRAS3, PLAGL1, SGCE, and SLC38A4, tissue-specific differences were detected. In addition, we examined the imprinting status of candidate genes by quantitative allelic pyrosequencing. Samples were collected from Day 30 pregnancies generated from reciprocal crosses of Meishan and White Composite breeds, and single-nucleotide polymorphisms were identified in candidate genes. Imprinting was confirmed for DIRAS3, DLK1, H19, IGF2AS, NNAT, MEST, PEG10, PHLDA2, PLAGL1, SGCE, and SNORD107. We also found no evidence of imprinting in ASB4, ASCL2, CD81, COMMD1, DCN, DLX5, and H13. Combined, these results represent the most comprehensive survey of imprinted genes in swine to date.

Conservation of genomic imprinting at the XIST, IGF2, and GTL2 loci in the bovine.

Genomic imprinting is theorized to exist in all placental mammals and some marsupials; however, extensive comparative analysis of animals aside from humans and mice remains incomplete. Here we report conservation of genomic imprinting in the bovine at the X chromosome inactivation–specific transcript (XIST), insulin-like growth factor 2 (IGF2), and gene trap locus 2 (GTL2) loci. Coding single nucleotide polymorphisms (SNPs) between Bos gaurus and Bos taurus were detected at the XIST, IGF2, and GTL2 loci, which have previously been identified as imprinted in either humans, mice, or sheep. Expression patterns of parental alleles in F1 hybrids indicated preferential paternal expression at the XIST locus solely in the chorion of females, whereas analysis of the IGF2 and GTL2 loci indicated preferential paternal and maternal expression of alleles, respectively, in both fetal and placental tissues. Comparative sequence analysis of the XIST locus and adjacent regions suggests that repression of the maternal allele in the bovine is controlled by a different mechanism than in mice, further reinforcing the importance of comparative analysis of imprinting.

THE EFFECTS OF HUMAN LEUKEMIA INHIBITORY FACTOR (HLIF) AND CULTURE MEDIUM ON IN VITRO DIFFERENTIATION OF CULTURED PORCINE INNER CELL MASS (PICM)

SummaryIsolation and maintenance of porcine embryonic stem (pES) cells have been hindered by the inability to inhibit differentiation of the porcine inner cell mass (pICM) in vitro. Culture conditions currently in use have been developed from mouse ES cell culture and are not effective for maintaining the pICM. Optimizing culture conditions for the pICM is essential. We have developed a grading system to detect changes in the differentiation status of in vitro cultured pICM. Porcine ICMs (Day 7) were isolated by immunosurgery and cultured for 4 d in either Dulbecco’s modified Eagle’s medium (DMEM)-based medium (D medium) or DMEM/Ham’s F-10 (1:1)-based medium (D/H medium) with or without human Leukemia Inhibitory Factor (hLIF, 1000 iu/ml). Colonies were photographed daily for morphological analysis. pICMs were categorized into one of two types based on their morphological profile: type A, nonepithelial or type B, epithelial-like. Eight investigators evaluated pICM differentiation using standardized differentiation profiles. Each pICM series was graded on a scale of 1 (fully undifferentiated) to 5 (fully differentiated) for each time point. Differentiation was verified by alkaline phosphatase activity, cytokeratin staining, and scanning electron microscopy. Neither hLIF nor culture medium delayed differentiation of pICMs (P=0.08 and P=0.25, respectively). The grading system employed was an effective tool for detecting treatment effects on differentiation of the developing pICM. These results demonstrate that hLIF cannot significantly inhibit differentiation of the pICM, and is unlikely to assist in porcine ES cell isolation. Future experiments utilizing homologous cytokines may prove more beneficial.

The role of imprinted genes in fetal growth abnormalities

Epigenetics, and in particular imprinted genes, have a critical role in the development and function of the placenta, which in turn has a central role in the regulation of fetal growth and development. A unique characteristic of imprinted genes is their expression from only one allele, maternal or paternal and dependent on parent of origin. This unique expression pattern may have arisen as a mechanism to control the flow of nutrients from the mother to the fetus, with maternally expressed imprinted genes reducing the flow of resources and paternally expressed genes increasing resources to the fetus. As a result, any epigenetic deregulation affecting this balance can result in fetal growth abnormalities. Imprinting-associated disorders in humans, such as Beckwith-Wiedemann and Angelman syndrome, support the role of imprinted genes in fetal growth. Similarly, assisted reproductive technologies in animals have been shown to affect the epigenome of the early embryo and the expression of imprinted genes. Their role in disorders such as intrauterine growth restriction appears to be more complex, in that imprinted gene expression can be seen as both causative and protective of fetal growth restriction. This protective or compensatory effect needs to be explored more fully.