Global genome analysis reveals a vast and dynamic anellovirus landscape within the human virome.

Anelloviruses are a ubiquitous component of healthy human viromes and remain highly prevalent after being acquired early in life. The full extent of "anellome" diversity and its evolutionary dynamics remain unexplored. We employed in-depth sequencing of blood-transfusion donor(s)-recipient pairs coupled with public genomic resources for a large-scale assembly of anellovirus genomes and used the data to characterize global and personal anellovirus diversity through time. The breadth of the anellome is much greater than previously appreciated, and individuals harbor unique anellomes and transmit lineages that can persist for several months within a diverse milieu of endemic host lineages. Anellovirus sequence diversity is shaped by extensive recombination at all levels of divergence, hindering traditional phylogenetic analyses. Our findings illuminate the transmission dynamics and vast diversity of anelloviruses and set the foundation for future studies to characterize their biology.

Adeno-associated virus (AAV)-mediated transduction of male germ line stem cells results in transgene transmission after germ cell transplantation.

We explored whether exposure of mammalian germ line stem cells to adeno-associated virus (AAV), a gene therapy vector, would lead to stable transduction and transgene transmission. Mouse germ cells harvested from experimentally induced cryptorchid donor testes were exposed in vitro to AAV vectors carrying a GFP transgene and transplanted to germ cell-depleted syngeneic recipient testes, resulting in colonization of the recipient testes by transgenic donor cells. Mating of recipient males to wild-type females yielded 10% transgenic offspring. To broaden the approach to nonrodent species, AAV-transduced germ cells from goats were transplanted to recipient males in which endogenous germ cells had been depleted by fractionated testicular irradiation. Transgenic germ cells colonized recipient testes and produced transgenic sperm. When semen was used for in vitro fertilization (IVF), 10% of embryos were transgenic. Here, we report for the first time that AAV-mediated transduction of mammalian germ cells leads to transmission of the transgene through the male germ line. Equally important, this is also the first report of transgenesis via germ cell transplantation in a nonrodent species, a promising approach to generate transgenic large animal models for biomedical research.

Production of recombinant albumin by a herd of cloned transgenic cattle.

Purified plasma derived human albumin has been available as a therapeutic product since World War II. However, cost effective recombinant production of albumin has been challenging due to the amount needed and the complex folding pattern of the protein. In an effort to provide an abundant source of recombinant albumin, a herd of transgenic cows expressing high levels of rhA in their milk was generated. Expression cassettes efficiently targeting the secretion of human albumin to the lactating mammary gland were obtained and tested in transgenic mice. A high expressing transgene was transfected in primary bovine cell lines to produce karyoplasts for use in a somatic cell nuclear transfer program. Founder transgenic cows were produced from four independent cell lines. Expression levels varying from 1-2 g/l to more than 40 g/l of correctly folded albumin were observed. The animals expressing the highest levels of rhA exhibited shortened lactation whereas cows yielding 1-2 g/l had normal milk production. This herd of transgenic cattle is an easily scalable and well characterized source of rhA for biomedical uses.

Effects of long-term administration of high-dose recombinant human antithrombin in immunosuppressed primate recipients of porcine xenografts.

BACKGROUND: Fibrin deposition is central to the acute humoral rejection process occurring in the presence of consumptive coagulopathy when pig organs are transplanted into primates. METHODS: To assess whether strategies aimed at preventing fibrin formation may extend xenograft survival, we administered high daily doses of recombinant human antithrombin (rhAT) (500 U/kg twice daily) to obtain both anticoagulant and anti-inflammatory effects in immunosuppressed primate recipients of porcine kidneys. RESULTS: Some degree of consumptive coagulopathy developed in both rhAT-treated (n=3) and untreated (n=3) primates. No major differences in the coagulation parameters analyzed were observed between the 2 groups. Similarly, no difference in survival was seen between rhAT-treated (20.6+/-4 days; range: 15-23 days) and untreated animals (17.3+/-11.6 days; range: 7-30 days), although the rhAT-treated primates had a higher bleeding tendency. Despite the high daily dose of rhAT, considerable fibrin deposition was observed in the graft as early as 2 weeks after transplantation. CONCLUSIONS: These results suggest that a high daily dose of rhAT fails to influence survival or prevent fibrin formation and deposition in the graft in our pig-to-primate model. However, the potential role of rhAT administered in combination with heparins or other clotting inhibitor concentrates in this model remains to be determined.

Endocrine profiles and growth patterns of cloned goats.

Two groups of goats produced by fetal somatic cell nuclear transfer (NT) were monitored to evaluate the similarities in growth patterns among cloned animals. Clone group I consisted of five Toggenburg females cloned from the same transgenic cell line and born to different recipient does. Clone group II consisted of two Saanen does born as twins to a single recipient female from a second transgenic cell line. Each cell line was constructed with a transgene (different for each clone group) that would express, producing a protein product in the milk during lactation of the does. Weight, hip height, and circulating levels of growth-related hormones were monitored at weekly and monthly intervals for comparison within the clone groups. A contemporary group (group III) consisting of seven crossbred does of similar ages and weights was also monitored for baseline endocrine values during the study. Serum samples from all groups were analyzed for growth hormone (GH), insulin-like growth factor I (IGF-I), triiodothyronine (T3), thyroxine (T4), and insulin via standard laboratory radioimmunoassay procedures. The averaged standard deviation from the mean was used to evaluate similarities within the groups of cloned does and the does in the contemporary group. The does in clone group II were less variable than the goats in clone group I for weight and hip height. This was perhaps due to a recipient effect. The two groups of cloned females were less variable than the contemporary group for circulating IGF-I and T4 concentrations. In contrast, the two groups of cloned does had at least one cloned group that was more variable than the contemporary group for GH, T3, and insulin. The animal variation, measured by the average standard deviation from the mean, of the cloned does is possibly due to environmental effects encountered in utero and/or in the postnatal period, as well as possible mitochondrial DNA differences between the cell line and donor oocytes used for NT. The variation among cloned does in this study indicates that the use of somatic cell NT to reproduce identical phenotypes may not succeed in all situations.

Effect of genetic background on glycosylation heterogeneity in human antithrombin produced in the mammary gland of transgenic goats.

Glycosylation is involved in the correct folding, targeting, bioactivity and clearance of therapeutic glycoproteins. With the development of transgenic animals as expression systems it is important to understand the impact of different genetic backgrounds and lactations on glycosylation. We have evaluated the glycosylation of recombinant antithrombin produced in several transgenic goat lines, from cloned animals and from different types of lactation including induced lactations. Our results show glycosylation patterns from the protein expressed in animals, derived from the same founder goat, are mostly comparable. Furthermore, the protein expressed in two cloned goats had highly consistent oligosaccharide profiles and similar carbohydrate composition. However, there were significantly different oligosaccharide profiles from the proteins derived from different founder goats. Artificial induction of lactation did not have significant effects on overall carbohydrate structures when compared to natural lactation. The only major difference was that recombinant antithrombin from induced lactations contained a slightly higher ratio of N-acetylneuraminic acid to N-glycolylneuraminic acid and less amount of oligosaccharides containing N-glycolylneuraminic acid. The oligosaccharides from all animals were a mixture of high mannose-, hybrid- and complex-type oligosaccharides. Sialic acid was present as alpha-2,6-linkage and no alpha-1,3-linked galactose was observed. These results indicate that transgenic animals with closely related genetic backgrounds express recombinant protein with comparable glycosylation.

Depletion of endogenous germ cells in male pigs and goats in preparation for germ cell transplantation.

The efficiency of germ cell transplantation, the procedure of transferring germ cells from a donor male into the testes of recipient males, can be greatly increased by reduction of endogenous germ cells in recipient animals. To develop effective methods for suppression of endogenous spermatogenesis in potential pig and goat recipients, we either administered busulfan to pregnant sows or irradiated the testes of immature goats. Piglets from sows treated twice with busulfan (7.5 mg/kg) at days 98 and 108 of gestation showed reduced gonocyte numbers at 2, 4, and 8 weeks of age and reduced initiation of spermatogenesis at 16 weeks of age. For goats, groups of 3 kids at 1, 5, or 9.5 weeks of age received fractionated irradiation of the testes with 3 doses of 2 Gy on 3 consecutive days. At 2 months after irradiation, 5%-10% of seminiferous tubule cross sections contained pachytene spermatocytes, compared with 50%-100% in controls. At 3 months after irradiation, spermatozoa appeared in 20% of tubule cross sections in all treated goats and in 100% of tubules in control goats. By 6 months after irradiation, spermatogenesis had recovered in 60% of tubules in goats treated at 5 or 9.5 weeks of age but in only 29% of tubules after treatment at 1 week of age. Therefore, late gestation in utero treatment of pigs with low doses of busulfan and testicular irradiation of goats at 1 week of age will result in a reduction in the endogenous germ cell population that could facilitate donor cell colonization after germ cell transplantation.

Effect of serum concentration, method of trypsinization and fusion/activation utilizing transfected fetal cells to generate transgenic dairy goats by somatic cell nuclear transfer.

This work was performed within a commercial nuclear transfer program to investigate different methods for synchronizing donor cell cycle stage, for harvesting donor cells, and for fusion and activation of reconstructed caprine embryos. Primary fetal cells isolated from day 35 to day 40 fetuses were co-transfected with DNA fragments encoding both the heavy and light immunoglobulin chains of three different monoclonal antibodies and neomycin resistance. Four neomycin resistant cell lines for each antibody were selected, expanded, and aliquots were both cryopreserved for later use as karyoplast donors or used for further genetic characterization. Transfected fetal cells were cultured in 0.5% FBS to synchronize G0/G1 cell cycle stage cells, then re-fed with 10% FBS prior to use to allow donor cells to re-enter the cell cycle. Alternatively, transfected fetal cells were grown to confluence in 10% FBS to induce contact inhibition to synchronize G0/G1 cell cycle stage cells. Adherent monolayers of transfected fetal donor cells were harvested by either partial or complete trypsinization. Donor cells were simultaneously fused and activated with enulceated in vivo produced ovulated oocytes from superovulated does. Half of the fused couplets received an additional electrical activation pulse and non-fused couplets were re-fused. Four live offspring were produced from 587 embryos generated from cell lines cultured in 0.5% FBS, while one live offspring was produced from 315 embryos generated from cell lines cultured in 10% FBS (0.7% versus 0.3% embryos transferred, respectively, P > 0.05). Five offspring were produced from 633 embryos generated from cell lines harvested by partial trypsinization (0.8% embryos transferred), and no offspring were produced from 269 embryos generated from cell lines harvested by complete trypsinization. Four live offspring were produced from 447 embryos generated from re-fused couplets, and one live offspring was produced from 230 embryos generated from fused couplets that received an additional electrical activation pulse (0.9% versus 0.4% embryos transferred, respectively, P > 0.05). These results suggest that low-serum culture of transfected goat fetal cells and harvest by partial trypsinization may be more efficient methods for generating transgenic goats by somatic cell nuclear transfer. In addition, re-fusion of non-fused couplet or an additional activation step was successful for producing live offspring.

Synchronization of goat fibroblast cells at quiescent stage and determination of their transition from G0 to G1 by detection of cyclin D1 mRNA.

A number of studies have reported that donor cells consisting of serum starved cells, which are assumed to be at quiescence (G0), or non-starved confluent cells or mitotic cells obtained by shake-off, both of which are assumed to be at G1 phase, give better results in nuclear transfer (NT) than cells at other phases of the cell cycle. Whether G0 or G1 cells function better as donor cells is yet to be determined by detailed studies. The aims of this study were to analyze the cell cycle of goat transfected fibroblasts and determine the timing of transition from G0 to G1 by detecting G1-specific marker, cyclin D1 mRNA. Fluorescent-activated cell sorting (FACS) analyses of cells after 4 days of serum starvation showed that more that 90% of cells were in G0/G1. Additionally, detection of cyclin D1 mRNA by northern blot analysis showed that 4-day serum starved quiescent cells started entering G1 a few hours after addition of 10% serum to the medium. Taken together, the data indicated that serum starved transfected primary fibroblasts of adult goats experienced the G0 to G1 transition within 5 h of serum stimulation and were at the mid-G1 stage within 10 h of serum stimulation.

Metabolism, protein content, and in vitro embryonic development of goat cumulus-oocyte complexes matured with physiological concentrations of glucose and L-lactate.

No information is available concerning how the maturation environment controls the metabolism of goat oocytes. The objectives of this experiment were to: (1) Determine the concentrations of glucose, lactate, and pyruvate in caprine follicular fluid; and (2) Investigate the effects of physiological concentrations of glucose and lactate in the in vitro maturation (IVM) medium on the metabolism (glycolysis and pyruvate oxidation), protein content, and developmental competence of caprine oocytes and cumulus-oocyte complexes (COCs). Abattoir-derived COCs were matured for 18-20 hr in a defined, SOF-based medium containing 0.75, 1.5 (follicular fluid = 1.4 mM), or 3.0 mM glucose, and 3.0, 6.0 (follicular fluid = 7.1 mM), or 12.0 mM L-lactate. The protein content of oocytes and COCs was not affected (P > 0.05) by the concentration of glucose and lactate in the maturation medium. Increasing glucose and lactate decreased (P < or = 0.05) glycolytic activity of oocytes, without affecting (P > 0.05) pyruvate oxidation. In COCs, increasing glucose concentrations tended (P = 0.07) to decrease glycolysis. When metabolic activity was corrected for protein content (pmol/microg protein/3 hr), increasing glucose or lactate concentrations in the medium decreased (P < or = 0.05) pyruvate oxidation in oocytes, but increased (P < or = 0.05) pyruvate oxidation in COCs. Embryonic development (cleavage and blastocyst development, hatching, and cell number) was not affected (P > 0.05) by the glucose and lactate concentrations tested. These results indicate that concentrations of glucose and lactate in the medium have cell type-specific effects on metabolism of oocytes and COCs, but do not affect developmental competence within the range of concentrations tested.

Fertility and germline transmission of donor haplotype following germ cell transplantation in immunocompetent goats.

Transplantation of spermatogonial stem cells into syngeneic or immunosuppressed recipient mice or rats can result in donor-derived spermatogenesis and fertility. Recently, this approach has been employed to introduce a transgene into the male germline. Germ-cell transplantation in species other than laboratory rodents, if successful, holds great promise as an alternative to the inefficient methods currently available to generate transgenic farm animals that can produce therapeutic proteins in their milk or provide organs for transplantation to humans. To explore whether germ-cell transplantation could result in donor-derived spermatogenesis and fertility in immunocompetent recipient goats, testis cells were transplanted from transgenic donor goats carrying a human alpha-1 antitrypsin expression construct to the testes of sexually immature wild-type recipient goats. After puberty, sperm carrying the donor-derived transgene were detected in the ejaculates of two out of five recipients. Mating of one recipient resulted in 15 offspring, one of which was transgenic for the donor-derived transgene. This is the first report of donor cell-derived sperm production and transmission of the donor haplotype to the next generation after germ-cell transplantation in a nonrodent species. Furthermore, these results indicate that successful germ-cell transplantation is feasible between immunocompetent, unrelated animals. In the future, transplantation of genetically modified germ cells may provide a more efficient alternative for production of transgenic domestic animals.

Loss of Emx2 function leads to ectopic expression of Wnt1 in the developing telencephalon and cortical dysplasia.

Leptomeningeal glioneuronal heterotopias are a focal type of cortical dysplasia in which neural cells migrate aberrantly into superficial layers of the cerebral cortex and meninges. These heterotopias are frequently observed as microscopic abnormalities in the brains of individuals with central nervous system (CNS) malformations and epilepsy. Previous work has demonstrated that the function of Emx2, which encodes a homeodomain transcription factor, is essential for development of the cortical preplate, which gives rise to the marginal zone and subplate. However, transcriptional targets of EMX2 during CNS development are unknown. We report that leptomeningeal glioneuronal heterotopias form in Emx2(-/-) mice that are equivalent to human lesions. Additionally, we observed ectopic expression of Wnt1 in the embryonic roofplate organizer region and dorsal telencephalon. To determine the phenotypic consequences of such Wnt1 misexpression, we deleted a putative EMX2 DNA-binding site from the Wnt1 enhancer and used this to misexpress Wnt1 in the developing murine CNS. Heterotopias were detected in transgenic mice as early as 13.5 days postcoitum, consistent with a defect of preplate development during early phases of radial neuronal migration. Furthermore, we observed diffuse abnormalities of reelin- and calretinin-positive cell populations in the marginal zone and subplate similar to those observed in Emx2-null animals. Taken together, these findings indicate that EMX2 is a direct repressor of Wnt1 expression in the developing mammalian telencephalon. They further suggest that EMX2-Wnt1 interactions are essential for normal development of preplate derivatives in the mammalian cerebral cortex.

Viable transgenic goats derived from skin cells.

The current study was undertaken to evaluate the possibility of expanding transgenic goat herds by means of somatic cell nuclear transfer (NT) using transgenic goat cells as nucleus donors. Skin cells from adult, transgenic goats were first synchronized at quiescent stage (G0) by serum starvation and then induced to exit G0 and proceed into G1. Oocytes collected from superovulated donors were enucleated, karyoplast-cytoplast couplets were constructed, and then fused and activated simultaneously by a single electrical pulse. Fused couplets were either co-cultured with oviductal cells in TCM-199 medium (in vitro culture) or transferred to intermediate recipient goat oviducts (in vivo culture) until final transfer. The resulting morulae and blastocysts were transferred to the final recipients. Pregnancies were confirmed by ultrasonography 25-30 days after embryo transfer. In vitro cultured NT embryos developed to morulae and blastocyst stages but did not produce any pregnancies while 30% (6/20) of the in vivo derived morulae and blastocysts produced pregnancies. Two of these pregnancies were resorbed early in gestation. Of the four recipients that maintained pregnancies to term, two delivered dead fetuses 2-3 days after their due dates, and two recipients gave birth to healthy kids at term. Fluorescence in situ hybridization (FISH) analysis confirmed that both kids were transgenic and had integration sites consistent with those observed in the adult cell line.