Components of the insulin-like growth factor system in in vivo - and in vitro-derived fetuses of cattle, and the association with growth and development.

This experiment was designed to study mechanisms affecting growth of in vivo-derived (IVD) and in vitro-produced (IVP) fetuses of cattle. Day-7 IVD or IVP cattle blastocysts were transferred to recipients, with pregnant females being slaughtered on Days 90 or 180 of gestation or allowed to undergo parturition. Uteri and contents were dissected and physically measured, and maternal and fetal plasma and amniotic and allantoic fluids were collected for IGF-1 and IGF-2 determinations, and IGFBP profile characterization. Transcripts for IGF-1 and IGF-2 mRNA in placental and fetal tissues, and IGF-1r and IGF-2r in placentomes were determined. There was a greater fetal weight in the IVP group, which was associated with greater IGF-1 and IGF-2 concentrations in maternal circulation, and changes in IGFBP profiles within fetal fluids. Day-90 IVP-derived fetuses were longer, had greater organ weights, larger placentomes, less placentome IGF-2r mRNA transcript, and greater maternal IGF-1 and IGF-2 concentrations than controls. On Day 180 and at parturition tissues from IVP-derived fetuses/calves were from larger uteri, with larger placentomes/fetal membranes, fetuses/calves weighed more, had greater fetal hepatic IGF-2 mRNA transcript, had less fetal plasma IGF-1 and greater allantoic IGF-2 concentrations, greater and lesser IGFBP activities in the allantoic and amniotic fluids, respectively, and greater glucose and fructose accumulation in fetal fluids. Components of the IGF system were differentially regulated not only according to the gestation period (Days 90 or 180) and fluid type (maternal or fetal plasma, amniotic or allantoic fluids), but also based on conceptus origin (IVP or IVD) in cattle.

Derivation of porcine pluripotent stem cells for biomedical research.

Pluripotent stem cells including embryonic stem cells (ESCs), embryonic germ cells (EGCs), and induced pluripotent stem cells (iPSCs) are capable of self-renew and limitlessly proliferating in vitro with undifferentiated characteristics. They are able to differentiate in vitro, spontaneously or responding to suitable signals, into cells of all three primary germ layers. Consequently, these pluripotent stem cells will be valuable sources for cell replacement therapy in numerous disorders. However, the promise of human ESCs and EGCs is cramped by the ethical argument about destroying embryos and fetuses for cell line creation. Moreover, there are still carcinogenic risks existing toward the goal of clinical application for human ESCs, EGCs, and iPSCs. Therefore, a suitable animal model for stem cell research will benefit the further development of human stem cell technology. The pigs, on the basis of their similarity in anatomy, immunology, physiology, and biochemical properties, have been wide used as model animals in the study of various human diseases. The development of porcine pluripotent stem cell lines will hold the opportunity to provide an excellent material for human counterpart to the transplantation in biomedical research and further development of cell-based therapeutic strategy.

Indistinguishable transcriptional profiles between in vitro- and in vivo-produced bovine fetuses.

During the past several decades, in vitro fertilization (IVF) has been increasingly used both in animal production and human infertility treatment. Animals derived from in vitro manipulation are occasionally associated with abnormal offspring syndrome (AOS) and other developmental abnormalities. By studying gene expression of in vitro-produced (IVP) embryos/animals, we gain an indicator of how well this procedure mimics the in vivo environment. Most previous studies of this nature have focused on only a few genes at a time or have been limited to studying the pre-implantation stage; a global view of how gene transcription may be influenced by in vitro procedures during fetal development has yet to be ascertained. To this end, we collected liver and placental tissue samples from IVP and in vivo control bovine fetuses at days 90 and 180 of gestation. We used a bovine 13K oligonucleotide microarray to investigate the transcriptional profiles in both tissues from IVP fetuses, and compared them with those of their age-matched in vivo counterparts. Surprisingly, in both liver and placental tissues, the transcriptional profiles between IVP and control fetuses, at either 90 or 180 days of gestation, were indistinguishable. A total of 879 genes were found to be significantly regulated during liver development from 90 to 180 days of gestation, but there were no gene expression changes in the placental tissue during this developmental period. Quantitative real-time RT-PCR on 11 selected genes confirmed these results. Our results have certain implications for IVF technologies, both in agriculture and in human medicine.

Transplantation of young ovaries restored cardioprotective influence in postreproductive-aged mice.

The female cardioprotective advantage, present in mammals of a reproductively competent age, is lost during the transition to a postreproductive state. The role of reproductive hormones in this transition is most evident in women with premature ovarian failure, where reduced estrogen production has been associated with an increased incidence of early death from cardiovascular disease. Previously, we reported that postreproductive-aged mice that received young ovaries displayed an increased life span. Subsequent histopathological analysis suggested the presence of a cardioprotective effect associated with the restoration of ovarian influence. This restoration in postreproductive-aged mice produced a sharp decrease in evidence of significant cardiomyopathy at death, compared with sham-transplanted mice (36.0% vs. 73.3%, respectively). Within the intact transplant group, evidence of cardiomyopathy at death was decreased in mice that were reproductively cycling at the time of transplant, compared with acyclic mice (26.7% vs. 50.0%, respectively). This observation reflects the importance of timing in restoration of ovarian influence in this study. Transplantation of young ovaries to intact, postreproductive-aged female mice provided significant, long-term restoration of a cardioprotective benefit, similar to that previously present during a reproductively competent age. In these mice, restoration of ovarian influence through ovarian transplantation may, in effect, have postponed the advance of age-associated cardiomyopathy to a point where the disease did not reach a clinically relevant threshold during the lifetime of the recipients. These results offer support for previous clinical observations suggesting that hormone replacement therapy can produce divergent results if initiated during the perimenopausal period, compared with the postmenopausal ages.

Ovarian status influenced the rate of body-weight change but not the total amount of body-weight gained or lost in female CBA/J mice.

Previously we reported that prepubertally ovariectomized mice that received young, transplanted ovaries at a postreproductive age displayed a 40% increase in life expectancy. To study this increase in life expectancy in greater detail, prepubertally ovariectomized and ovary-intact CBA/J mice underwent ovarian transplantation at 11 months with 60-day-old ovaries or a sham surgery. Life span was significantly increased in transplant recipients. Body-weight changes of mice in each group were measured from the time of surgery (11 months) to death. Neither ovariectomy nor ovarian transplantation influenced the amount of peak body-weight attained or body-weight retained at death. However, the time (days) to peak body-weight was decreased by ovariectomy and ovarian transplant recipients displayed a trend toward an increase in time to peak weight. In addition, ovarian transplantation decreased the rate of weight loss to death. These results demonstrate that ovarian status, examined by means of ovariectomy and ovarian transplantation, clearly influenced the rate of weight change, but not the total amount of weight gain or loss in female mice.

Transplantation of young ovaries to old mice increased life span in transplant recipients.

Previously we reported that prepubertally ovariectomized mice that received young transplanted ovaries at a postreproductive age showed a 40% increase in life expectancy. To study this phenomenon in greater detail, 11-month-old ovariectomized and ovary-intact CBA/J mice underwent ovarian transplantation with 60-day-old ovaries or a sham surgery. Results from observations on transplant recipients in the current study extended our previous results. Whereas intact control mice lived an average of 726 days, transplant recipients lived an average of 770 days (i.e., 780 days for intact recipients and 757 days for ovariectomized recipients). If intact recipients had ceased reproductive cycling by the time of transplant, we observed a further increase in mean life span to 811 days. These results demonstrate that young ovaries enhanced longevity when transplanted to old mice and that ovarian status, examined by means of ovariectomy and ovarian transplantation, clearly influenced the potential of young transplanted ovaries to positively impact longevity.

Methylation changes in porcine primordial germ cells.

Epigenetic re-programming is an important event in the development of primordial germ cells (PGC) into functional gametes, characterized by genome-wide erasure of DNA methylation and re-establishment of epigenetic marks, a process essential for restoration of the potential for totipotency. In this study changes in the methylation status of centromeric repeats and two IGF2-H19 differentially methylated domain (DMD) sequences were examined in porcine PGC between Days 24 and 31 of pregnancy. The methylation levels of centromeric repeats and IGF2-H19 DMD sequences decreased rapidly from Days 24 to 28 in both male and female PGC. At Days 30 and 31 of pregnancy centromeric repeats and IGF2-H19 DMD sequences acquired new methylation in male PGC, while in female PGC these sequences were completely demethylated by Day 30 and remained hypomethylated at Day 31. To characterize methylation changes that PGC undergo in culture, the methylation status of embryonic germ cells (EGCs) derived from PGC at Day 26 of pregnancy was examined. Centromeric repeats and IGF2-H19 DMD sequences were similarly methylated in both male and female EGC and hypermethylated in female EGC compared with female PGC at the same embryonic age. Our results show that, similar to murine PGC, porcine PGC undergo genome-wide DNA demethylation shortly after arrival in the genital ridges. When placed in culture porcine PGC terminate their demethylation program and may acquire new DNA methylation marks. To our knowledge, this is the first report regarding epigenetic re-programming of genital ridge PGC in the pig.

Culture of porcine embryonic germ cells in serum-supplemented and serum-free conditions: the effects of serum and growth factors on primary and long-term culture.

Fetal bovine serum (FBS) is a commonly used medium supplement with variable and undefined composition, which presents problems in culture of pluripotent stem cells. The purpose of this study was to determine if FBS can be replaced with Knockout Serum Replacement (KSR), a defined medium supplement, and to examine the effects of FBS and growth factors on short- and long-term culture of pig embryonic germ cells (EGC). No significant differences were observed in total and mean colony areas in primary cultures between FBS- and KSR-supplemented medium (421 x 10(3) mum(2) vs. 395 x 10(3) microm(2), p = 0.68, n = 11, and 6375 microm(2) vs. 6407 microm(2), p = 0.885, respectively). Total and mean colony areas were significantly larger in KSR-supplemented medium compared with medium supplemented with KSR and growth factors (505 x 10(3) microm(2) vs. 396 x 10(3) microm(2), p = 0.016, n = 12, and 8769 microm(2) vs. 6513 microm(2), p = 0.003, respectively). The cultures proliferated for significantly higher numbers of passages in FBS-supplemented medium and in medium supplemented with KSR and growth factors compared with medium containing KSR alone (31.1 vs. 21.9, p = 0.004, n = 10, and 35.5 vs. 21.6, p = 002, n = 10, respectively). Porcine EGC maintained in serum-free conditions were positive for pluripotent stem cell markers, maintained stable karyotypes for up to 54 passages, and were capable of differentiating in vitro into cells from the three primary germ layers. These results will help improve and standardize culture of pluripotent stem cells in the pig.

Angiogenesis in day-30 bovine pregnancies derived from nuclear transfer.

Impaired placental angiogenesis during early pregnancy may result in placental defects that adversely affect development of nuclear-transfer (NT) embryos later in pregnancy. These experiments were designed to quantify and compare development of placental microvasculature and expression of genes associated with angiogenesis, including members of the VEGF and angiopoietin (Ang) families, in maternal and embryonic placental tissues of day 30 bovine concepti derived from NT or in vitro fertilization (IVF) followed by in vivo development to the blastocyst stage in the sheep oviduct. Microvascular volume density (MVD) within the caruncular tissues, as determined using Periodic Acid-Schiff's staining as well as immunohistochemical staining for von Willebrand's factor, was not different between NT- and IVF- derived pregnancies. Expression of genes implicated in angiogenic mechanisms, including VEGF-A and -C, placental growth factor (PlGF), VEGF receptors (Flt-1, Flk-1, and Flt-4), angiopoietin-1 (Ang1), Ang2, Tie1, Tie2, and hypoxia-inducible factor (HIF), were determined. In chorio-allantoic membranes, levels of PlGF transcripts were significantly lower in NT- than IVF-derived tissues (p<0.05), whereas HIF-1alpha transcription in chorio-allantoic membranes of cloned concepti was higher at p<0.10. Caruncular expression of HIF-1alpha and Ang1 also was increased in NT-derived pregnancies at p

Perinatal physiology in cloned and normal calves: physical and clinical characteristics.

The period immediately after birth is a vital time for all newborn calves as the cardiovascular, respiratory, and other organ systems adapt to life ex utero. Reported neonatal mortality rates suggest this period to be especially critical in cloned calves; yet prospective, controlled studies on the physiological status of these calves are lacking. The objectives of this study were to compare neonatal (birth to 48 h of age) physical and clinical characteristics and placental morphology of cloned and embryo transfer control calves delivered by cesarean section after induced labor. All calves were raised under specialized neonatal-care protocols at a large-animal veterinary research and teaching hospital. Cloned calves were similar to controls for many parameters studied. Notable exceptions included developmental delays of important physical adjustment parameters and enlargement of the umbilical region. Placentas associated with cloned calves contained fewer total placentomes, a twofold increase in surface area and mass per placentome, and a shift in placentome morphology toward larger, flatter placentomes. The most striking clinical variations detected in clones were hypoglycemia and hyperfructosemia, both measures of carbohydrate metabolism. Because the placenta is known to be the source of plasma fructose in newborn calves, increased fructose production by the cloned placenta may be an important factor in the etiology of umbilical and cardiac anomalies in clones observed in this and other studies.

Perinatal physiology in cloned and normal calves: hematologic and biochemical profiles.

Although a majority of clones are born normal and apparently healthy, mortality rates of nearly 30% are described in many reports. Such losses are a major limitation of cloning technology and represent substantial economic investment as well as justifiable animal health and welfare concerns. Prospective, controlled studies are needed to understand fully the causes of neonatal mortality in clones and to develop preventive and therapeutic strategies to minimize losses. We report here the findings of studies on the hematologic and biochemical profiles of cloned and control calves in the immediate 48-h postpartum period. Cloned calves were similar to control calves for a majority of parameters studied including blood gases, concentrations of plasma proteins, minerals and electrolytes, and white blood cell, neutrophil, lymphocyte, and platelet counts. The most notable differences between clones and controls in this study were reduced red- and white-blood cell counts in clones at birth and 1 h of age. As a group, plasma electrolyte concentrations were more variable in clones, and the variability tended to be shifted either higher (sodium, chloride) or lower (potassium, bicarbonate) than in controls. Previously, we noted differences in carbohydrate parameters, the length of time required for clones to make the neonatal adaptation to life ex utero, and morphology of the cloned placenta. Taken together, our findings suggest that cloned calves experience greater difficulty adjusting to life ex utero and that further research is warranted to determine the nature of the relationship between the physiological differences noted here in clones at birth and concomitant abnormal placental morphology.

Transient depletion of Ku70 and Xrcc4 by RNAi as a means to manipulate the non-homologous end-joining pathway.

Non-homologous end joining (NHEJ) is the major DNA double-strand break (DSB) repair pathway in mammalian cells and is likely responsible for the non-homologous integration of transgenes. In higher eukaryotes, this pathway predominates over the homologous recombination (HR) pathway and therefore may account for the low level of HR events that occur in mammalian cells. We evaluated the effects of transient RNAi-induced down-regulation of key components of the NHEJ pathway in human HCT116 cells. Treatment with siRNA targeting Ku70 and Xrcc4 reduced corresponding protein levels by 80-90% 48h after transfection, with a return to normal levels by 96h. Additionally, down-regulation of Ku70 and Xrcc4 resulted in a concomitant depletion of both Ku70 and Ku86 proteins. Biological consequences of transient RNAi-mediated depletion of Ku70 and Xrcc4 included sensitization to gamma radiation and a significant decrease in the expression of a linear GFP reporter gene. The results highlight the possibility of a successful means to manipulate the NHEJ pathway by RNAi.

The effect of coating single- and double-stranded DNA with the recombinase A protein of Escherichia coli on transgene integration in mice.

Embryo survival and transgene integration rates are two major factors that influence the efficiency of transgenic animal production by pronuclear microinjection. Recombinase A protein-coated transgenes were compared for transgene integration and embryo survival with their non-coated counterparts in both single- and double-stranded forms. Murine zygotes were microinjected with a large 30 kb alpha(S1)-casein/human lysozyme DNA construct and a small 5.5 kb beta-lactoglobulin/desaturase DNA construct using four different construct preparations for each gene. The preparations included recombinase A protein-coated, single- and double-stranded DNA constructs and non-coated, single- and double-stranded DNA constructs. Using conventional non-coated, double-stranded DNA constructs, we obtained a transgene integration efficiency of 1.5% (1352 embryos transferred produced 20 transgenic pups). The same double-stranded DNA constructs coated with recombinase A protein yielded a similar percentage of transgene integration (1.1%, 18/1697). Using single-stranded DNA, non-coated constructs produced a transgene integration rate of 0.5%, while none of the 1040 zygotes injected with recombinase A-coated constructs produced transgenic pups. While recombinase A protein coating produced no effect on embryo survival, litter size or pregnancy rate with double-stranded constructs, a detrimental effect was observed on embryo survival (P < 0.001) and pregnancy rate (P < 0.005) with recombinase A protein coating of single-stranded human lysozyme DNA constructs. A trend toward increased embryo survival (P = 0.054) with no difference in pregnancy rate (P > 0.05) was observed with the recombinase A protein coating of single-stranded desaturase constructs. These results suggest that recombinase A protein coating of single- and double-stranded DNA constructs produced no significant differences (P > 0.05) in the efficiency of generating transgenic mice with respect to the percentage of transgenic animals born.

Consumption of milk from transgenic goats expressing human lysozyme in the mammary gland results in the modulation of intestinal microflora.

Lysozyme is a key antimicrobial component of human milk that has several health-promoting functions including the development of a healthy intestinal tract. However, levels of lysozyme in the milk of dairy animals are negligible. We have generated transgenic dairy goats that express human lysozyme (HLZ) in their milk in an attempt to deliver the benefits of human milk in a continual fashion. To test the feasibility of this transgenic approach to achieve a biological impact at the level of the intestine, feeding trials were conducted in two animal models. Pasteurized milk from HLZ transgenic animals was fed to both kid goats (ruminant model) and young pigs (human model), and the numbers of total coliforms and Escherichia coli present in the small intestine were determined. Data from this proof-of-principle study demonstrate that milk from transgenic animals was capable of modulating the bacterial population of the gut in both animal models. Pigs that consumed pasteurized milk from HLZ transgenic goats had fewer numbers of coliforms and E. coli in their intestine than did those receiving milk from non-transgenic control animals. The opposite effect was seen in goats. Milk from these transgenic animals not only represent one of the first transgenic food products with the potential of benefiting human health, but are also a unique model to study the development and role of intestinal microflora on health, well-being and resistance to disease.

Expression profile and protein levels of placental products as indirect measures of placental function in in vitro-derived bovine pregnancies.

Bovine conceptus development and its association with placental proteins present in maternal, foetal and neonatal plasma and foetal (amniotic and allantoic) fluids were investigated in in vivo- and in vitro-produced (IVP) concepti and newborn calves. Females were superovulated to obtain control embryos, whereas IVP embryos were derived from established in vitro procedures. Pregnant animals were slaughtered on days 90 or 180 of gestation or allowed to develop to term for the assessment of physical traits. Foetal, maternal and neonatal blood and foetal fluids were collected for the determination of bovine placental lactogen (bPL) and bovine pregnancy-specific protein B (bPSPB) concentrations. Placental transcripts for bPL and bPSPB, determined by quantitative RT-PCR, were elevated in IVP placentomes. No major physical differences were observed between groups on day 90, but concentrations of bPL and bPSPB were higher in foetal plasma and allantoic fluid of IVP concepti in day 180 pregnancies, which were correlated with larger uterine and conceptus traits. Maternal concentrations of bPL in IVP pregnancies were lower than controls during the last 8 weeks of gestation, to become similar as parturition approached. Newborn IVP calves and foetal membranes were larger and displayed higher concentrations of plasma bPL than controls (10 and 60 min after birth). Our results indicated that differential patterns of secretion of bPL and bPSPB into the maternal and foetal systems occurred at distinct stages of gestation, and these were associated with altered conceptus development after in vitro embryo manipulations, indirectly demonstrating deviations in placental function in IVP pregnancies.

Human lysozyme expressed in the mammary gland of transgenic dairy goats can inhibit the growth of bacteria that cause mastitis and the cold-spoilage of milk.

The addition of human milk components with intrinsic antimicrobial activity to livestock milk by genetic engineering has the potential to benefit milk safety and production as well as the health of the lactating animal. As a model for the dairy cow, we generated transgenic goats that expressed human lysozyme in their milk at 68% of the levels found in human milk. Milk from these transgenic animals had a bacteriostatic effect on both in vitro and in vivo growth of several microorganisms important to the dairy industry. In vitro, milk from transgenic animals was capable of slowing the growth of mastitis-causing strains of Escherichia coli (P < 0.02) and Staphylococcus aureus (P < 0.05) as well as the cold-spoilage organism Pseudomonas fragi (P < 0.02). The growth of an organism involved in cheese-making, Lactococcus lactis, was not affected by the presence of lysozyme in milk. The supplementation of control milk with purified lysozyme did not achieve the same inhibitory effect as milk from transgenic animals. In vivo, milk from transgenic animals supported less bacterial growth than control milk. This transgenic model demonstrates the possibilities offered by genetic engineering to enhance the antimicrobial nature of milk and the udder.

Effect of the nuclear-donor cell lineage, type, and cell donor on development of somatic cell nuclear transfer embryos in cattle.

Potential applications of somatic cell nuclear transfer to agriculture and medicine are currently constrained by low efficiency and high rates of embryonic, fetal, and neonatal loss. Nuclear transfer efficiency in cattle was compared between three donor-cell treatments from a single animal, between four donor-cell treatments in sequential stages of differentiation from a single cell lineage and genotype, and between the same cell type in two donors. Cumulus and granulosa donor cells resulted in a greater proportion of viable day-7 embryos than ear-skin cells; pregnancy rate and losses were not different among treatments. The least differentiated cell type in the follicular cell lineage, preantral follicle cells, resulted in fewer cloned blastocysts (11%) than cumulus (30%), granulosa (23%), and luteal (25%) donor cells. Cloned blastocysts that did develop from preantral follicle cells (75%) were more likely to progress through implantation into later stages of pregnancy than cloned blastocysts from cumulus (10%), granulosa (9%), and luteal (11%) donor cells (p < 0.05). Day-7 embryo development from granulosa cells was similar between two donors (19 vs. 24%) and proved to be a poor indicator of further development as day-30 pregnancy rates varied threefold between donors (48 vs. 15%, p < 0.05). Results reported here emphasize the crucial role of the nuclear donor cell in the outcome of the nuclear-transfer process.

Measures of maternal-fetal interaction in day-30 bovine pregnancies derived from nuclear transfer.

Embryonic mortality and abnormal placental morphology have been reported by most researchers studying nuclear transfer (NT), and it now is accepted that placental anomalies and poor development of cloned embryos are related. As early as day 50 of gestation, cloned bovine concepti exhibit poor structural organization of the developing placentomes. These experiments were designed to identify alterations in maternal-fetal interactions during establishment of the placentas of NT-derived embryos at day 30 of gestation. Bovine NT embryos were produced using cultured fibroblast cells from a single Hereford donor cow, and control embryos were derived from in vitro fertilization (IVF). Following in vivo culture in ligated sheep oviducts, day-8 blastocysts were transferred to synchronized recipient heifers. Tissues recovered from viable day-30 pregnancies were analyzed by real-time RT-PCR, immunohistochemistry, and quantitative histological techniques. Immunoperoxidase staining of caruncular tissue from NT- and IVF-derived pregnancies revealed no significant differences in expression of the extracellular matrix proteins, collagen type IV and laminin, or the receptor subunits, integrins alpha1 and alpha3, suggesting that altered expression of these proteins at day 30 of gestation is not a primary cause of abnormal placentome structure in cloned concepti. Percentage of binucleate cells (BNC) within the trophoblast also was similar in NT- and IVF-derived pregnancies; however, expression of the BNC-specific placental lactogen (PL) transcript was elevated in NT-derived concepti (p < 0.05). These results indicate that regulation of PL transcription was altered in cloned day-30 placental tissues, suggesting the presence of irregular fetal-maternal signaling patterns that might undermine continued development of NT-derived concepti.

Attempts to enhance production of porcine chimeras from embryonic germ cells and preimplantation embryos.

Porcine embryonic germ (EG) cells share common features with porcine embryonic stem (ES) cells, including morphology, alkaline phosphatase activity and capacity for in vitro differentiation. Porcine EG cells are also capable of in vivo development by producing chimeras after blastocyst injection; however, the proportion of injected embryos that yield a chimera and the proportion of cells contributed by the cultured cells in each chimera are too low for practical use in genetic manipulation. Moreover, somatic, but not germ-line chimerism, has been reported from blastocyst injection using porcine ES or EG cells. To test whether efficiency of chimera production from blastocyst injection can be improved upon by changing the host embryo, we used as host embryos four groups according to developmental stage or length in culture: fresh 4-cell and 8-cell stage embryos subsequently cultured into blastocysts, fresh morulae, fresh blastocysts, and cultured blastocysts. Injection and embryo transfer of fresh and cultured blastocysts produced similar percentages of live piglets (17% versus 19%). Four piglets were judged to have a small degree of pigmentation chimerism, but microsatellite analysis failed to confirm chimerism in these or other piglets. Polymerase chain reaction analysis for detection of the porcine SRY gene in female piglets born from embryos injected with male EG cells identified six chimeras, at least one, but not more than two, from each treatment. Chimerism was confirmed in two putative pigmentation chimeras and in four piglets without overt signs of chimerism. The low percentage of injected embryos that yielded a chimera and the small contribution by EG cells to development of each confirmed chimera indicated that procedural changes in how EG cells were combined with host embryos were unsuccessful in increasing the likelihood that porcine EG cells will participate in embryonic development. Alternatively, our results suggested that improvements are needed in EG cell isolation and culture procedures to ensure in vitro maintenance of EG cell developmental capacity.

Increased efficiency of transgenic livestock production.

Production of transgenic livestock by pronuclear microinjection of DNA into fertilized zygotes suffers from the compounded inefficiencies of low embryo survival and low integration frequencies of the injected DNA into the genome. These inefficiencies are one of the major obstacles to the large-scale use of pronuclear microinjection techniques in livestock. We investigated exploiting the properties of recombinase proteins that allow them to bind DNA to generate transgenic animals via pronuclear microinjection. In theory, the use of recombinase proteins has the potential to generate transgenic animals with targeted changes, but in practice we found that the use of RecA recombinase-coated DNA increases the efficiency of transgenic livestock production. The use of RecA protein resulted in a significant increase in both embryo survival rates and transgene integration frequencies. Embryo survival rates were doubled in goats, and transgene integration was 11-fold higher in goats and three-fold higher in pigs when RecA protein-coated DNA was used compared with conventional DNA constructs without RecA protein coating. However, a large number of the transgenic founders generated with RecA protein-coated DNA were mosaic. The RecA protein coating of DNA is straightforward and can be applied to any species and any existing microinjection apparatus. These findings represent significant improvements on standard pronuclear microinjection methods by enabling the more efficient production of transgenic livestock.