Establishment of goat embryonic stem cells from in vivo produced blastocyst-stage embryos.

Embryonic stem (ES) cells with the capacity for germ line transmission have only been verified in mouse and rat. Methods for derivation, propagation, and differentiation of ES cells from domestic animals have not been fully established. Here, we describe derivation of ES cells from goat embryos. In vivo-derived embryos were cultured on goat fetal fibroblast feeders. Embryos either attached to the feeder layer or remained floating and expanded in culture. Embryos that attached showed a prominent inner cell mass (ICM) and those that remained floating formed structures resembling ICM disks surrounded by trophectodermal cells. ICM cells and embryonic disks were isolated mechanically, cultured on feeder cells in the presence of hLIF, and outgrown into ES-like colonies. Two cell lines were cultured for 25 passages and stained positive for alkaline phosphatase, POU5F1, NANOG, SOX2, SSEA-1, and SSEA-4. Embryoid bodies formed in suspension culture without hLIF. One cell line was cultured for 2 years (over 120 passages). This cell line differentiated in vitro into epithelia and neuronal cells, and could be stably transfected and selected for expression of a fluorescent marker. When cells were injected into SCID mice, teratomas were identified 5-6 weeks after transplantation. Expression of known ES cell markers, maintenance in vitro for 2 years in an undifferentiated state, differentiation in vitro, and formation of teratomas in immunodeficient mice provide evidence that the established cell line represents goat ES cells. This also is the first report of teratoma formation from large animal ES cells.

In vitro and in vivo characterization of recombinant human butyrylcholinesterase (Protexia) as a potential nerve agent bioscavenger.

Previous studies in rodents and nonhuman primates have demonstrated that pretreatment with cholinesterases can provide significant protection against behavioral and lethal effects of nerve agent intoxication. Human butyrylcholinesterase (HuBuChE) purified from plasma has been shown to protect against up to 5 x LD50s of nerve agents in guinea pigs and non-human primates, and is currently being explored as a bioscavenger pretreatment for human use. A recombinant form of HuBuChE has been expressed in the milk of transgenic goats as a product called Protexia. Protexia was supplied by Nexia Biotechnologies (Que., Canada) as a purified solution with a specific activity of 600 U/mg. Initial in vitro studies using radiolabeled 3H-soman or 3H-DFP (diisopropyl fluorophosphate) demonstrated that these inhibitors specifically bind to Protexia. When Protexia was mixed with soman, sarin, tabun or VX using varying molar ratios of enzyme to nerve agent (8:1, 4:1, 1:1 and 1:4, respectively), the data indicated that 50% inhibition of enzyme activity occurs around the 1:1 molar ratio for each of the nerve agents. Protexia was further characterized for its interaction with pyridostigmine bromide and six unique carbamate inhibitors of cholinesterase. IC50 and Ki values for Protexia were determined to be very similar to those of HuBuChE purified from human plasma. These data suggest that Protexia has biochemical properties very similar to those HuBuChE when compared in vitro. Together these data the continued development of the goat milk-derived recombinant HuBuChE Protexia as a potential bioscavenger of organophosphorus nerve agents.

State of the art in the production of transgenic goats.

This review summarises recent advances in the field of transgenic goats for the purpose of producing recombinant proteins in their milk. Production of transgenic goats via pronuclear microinjection of DNA expression vectors has been the traditional method, but this results in low efficiencies. Somatic cell nuclear transfer has dramatically improved efficiencies in rates of transgenesis. Characterisation of transfected cells in vitro before use in nuclear transfer guarantees that kids born are transgenic and of predetermined gender. Using these platform technologies, several recombinant proteins of commercial interest have been produced, although none of them has yet gained marketing approval. Before these technologies are implemented in goat improvement programmes, efficiencies must be improved, costs reduced, and regulatory approval obtained for the marketing of food products derived from such animals.

Advanced assisted reproduction technologies (ART) in goats.

Assisted reproduction technologies (ART) are reviewed with special emphasis on goat genetic improvement programs. Estrous synchronization and artificial insemination are the most commonly used ART worldwide because of their simplicity and excellent cost/benefit, especially when proven sires are used. Multiple ovulation and embryo transfer (MOET) has not become widely used due to its unpredictability. In vitro embryo production using oocytes collected by laparoscopy from valuable donors has the potential to improve the results obtained from MOET and expand its applications (for example, using prepubertal donors). However, the costs and inefficiencies of the system might restrict its use to special situations. Finally, transgenesis and cloning are expected to have a significant impact on the future genetic improvement of livestock. However, because of low efficiencies and high costs, their present use is restricted to applications with high returns such as the production of recombinant proteins of pharmaceutical and biomedical interest.

Prepubertal propagation of transgenic cloned goats by laparoscopic ovum pick-up and in vitro embryo production.

The use of laparoscopic ovum pick-up (LOPU) followed by in vitro embryo production was evaluated in the early propagation of cloned goats. Ten kinder goats produced by somatic cell nuclear transfer technology were used as oocyte donors. Half of the donor animals were subjected to LOPU at 2-3 months of age (prior to induction of lactation), whereas the other five goats were subjected to LOPU at 6-7 months of age (following induction to lactation). They were stimulated with 80 mg NIH-FSH-P1 (Folltropin, Vetrepharm, Canada) together with 300 IU eCG (Novormon, Vetrepharm, Canada) administered intramuscularly 36 h prior to LOPU. The number of follicles aspirated and oocytes recovered was higher in the younger group of donors (57 +/- 7 and 41 +/- 4 vs. 28 +/- 2 and 25.8 +/- 2, p < 0.05), however, oocytes from animals in the late prepubertal age showed higher developmental capacity resulting in higher transferable embryo yield (81.4% vs. 67.8%, p < 0.01), pregnancy rate (80% vs. 40%, p < 0.05) and total kids born (27 vs. 15, p < 0.01). In conclusion, LOPU in combination with in vitro embryo production techniques is an efficient method for the early propagation of valuable goats produced by somatic cell nuclear transfer.

Effects of repetition, interval between treatments and season on the results from laparoscopic ovum pick-up in goats.

The present study was conducted to evaluate the follicular response and oocyte yield following repeated gonadotrophin stimulation and laparoscopic aspiration in goats and to assess the effects of the time interval between procedures and season. A total of 98 adult goats were subjected to laparoscopic ovum pick-up (LOPU) five consecutive times in a transgenic production programme. Oestrus was synchronised by means of intravaginal sponges inserted for 10 days coupled with 125 microg cloprostenol 36 h before sponge removal and LOPU, and follicular development was stimulated with 80 mg follicle stimulating hormone and 300 IU equine chorionic gonadotrophin administered 36 h before LOPU. No difference was detected in the response for LOPUs 1, 2, 3 and 4. Although a small decrease in response was detected at LOPU 5 (P < 0.05), the numbers of follicles aspirated and oocytes recovered were not different from those at LOPU 1 and LOPUs 1 and 4, respectively. With respect to time interval between LOPU and season, all intervals and seasons produced acceptable responses, with no difference in follicles aspirated and oocytes recovered between intervals and seasons. These results indicate that LOPU may be repeated up to five times in goats at different intervals and in different seasons with little or no important change in overall response.

The in vitro and in vivo development of goat embryos produced by intracytoplasmic sperm injection using tail-cut spermatozoa.

The objective of this study was to assess the efficacy of a novel intracytoplasmic sperm injection (ICSI) procedure, as well as the in vitro and in vivo developmental competence of goat embryos produced by ICSI. Oocyte-cumulus complexes recovered by LOPU from donors stimulated with gonadotrophins were matured in vitro. Fresh goat semen was used for ICSI following Percoll gradient washing. Tail-cut spermatozoa were microinjected into the ooplasm of goat oocytes using a piezo micropipette-driving system (PiezoDrill). In order to assess developmental competence, the ICSI-derived zygotes were cultured in one of two media systems (mTALP-mKSOM vs G1.3-G2.3) for in vitro development or were transferred into recipients for full-term development. The results suggest that cutting sperm tails using the oocyte-holding pipette coupled with the PiezoDrill is an efficient approach for goat ICSI in terms of oocyte survival, pronuclear development and initial cleavage. The mTALP-mKSOM culture system was more suitable for in vitro development of ICSI-derived goat embryos than G1.3-G2.3. This first report of full-term development of an ICSI-derived goat embryo suggests that ICSI can be applied to assisted reproduction in goats.

Production of transgenic goats by pronuclear microinjection of in vitro produced zygotes derived from oocytes recovered by laparoscopy.

Oocytes collected by laparoscopic ovum pick-up (LOPU) were successfully used to produce transgenic goats by pronuclear microinjection of in vitro zygotes. Estrus cycles of 109 donor goats were synchronized using intravaginal sponges impregnated with 60 mg of medroxyprogesterone acetate and treatment with 70 mg NIH-FSH-P1 and 300 IU eCG to stimulate follicular development. Follicles were aspirated under laparoscopic observation. In vitro maturation (IVM) of oocytes was performed in M199 supplemented with hormones, kanamycin and 10% estrus goat serum. Following IVM, oocytes were cocultured with capacitated semen in TALP supplemented with 20% estrus goat serum for 15-20 h. The resulting zygotes were microinjected with a linear DNA fragment. In total, 3293 follicles were aspirated (15.7+/-9 follicles aspirated per donor) and 2823 oocytes were recovered (13.4+/-8 oocytes per donor). A total of 1366 zygotes were microinjected and transferred into 219 recipient goats by midventral laparotomy (average 6.2 embryos per recipient). A total of 150 kids were born, of which 9 (6 M: 3 F) were confirmed to be transgenic by PCR and Southern blotting analyses. These results demonstrate that acceptable transgenesis rates can be obtained in goats by DNA microinjection of in vitro produced zygotes.

Transgenic goats produced by DNA pronuclear microinjection of in vitro derived zygotes.

This study was undertaken to investigate various factors affecting the outcomes of in vitro fertilization (IVF) of oocytes retrieved by laparoscopic ovum pick-up (LOPU) technique from prepubertal and adult goats, as well as to evaluate the developmental competence of in vitro produced embryos. Oocyte-cumulus complexes recovered by LOPU from donors stimulated with gonadotrophins were matured in vitro. Fresh semen was used for IVF following various capacitation treatments. In vitro produced zygotes were either cultured to assess in vitro development or were transferred into recipients for full term development. The results indicated that successful IVF of the goat oocytes was affected by factors such as sperm capacitation treatment, oocyte quality, and abundance of cumulus cells on zona pellucida. Oocytes from both prepubertal and adult goats demonstrated similar full term developmental competence despite the fact that in vitro developmental rates were lower for prepubertal goats. The births of transgenic offspring demonstrated that the established LOPU-IVF technology combined with pronuclear microinjection can be successfully used to produce transgenic goats.

Advances in the production and propagation of transgenic goats using laparoscopic ovum pick-up and in vitro embryo production technologies.

Laparoscopic ovum pick-up (LOPU) is a convenient methodology by which oocytes can be recovered and used either for in vitro production of zygotes or as a source of cytoplasts in nuclear transfer (NT) procedures. The pregnancy and transgenesis rates achieved with IVM/IVF of LOPU-sourced oocytes followed by subsequent DNA microinjection of zygotes are similar to the rates obtained when using in vivo-produced oocytes or zygotes. Similarly, pregnancy rates and kids born by using LOPU-sourced and in vitro matured oocytes as recipient cytoplasts in NT programs are comparable with those reported by others using in vivo matured oocytes collected by oviduct flushing. The use of LOPU allows for improved control over the stage of maturation/development of the oocytes and produced zygotes, a less invasive means of recovery, thereby allowing for repeated usage of the oocyte donor animals and the ability to source the oocytes from live animals of known health status. In addition, because of large follicular responses that can be obtained from prepubertal animals, LOPU followed by IVM/IVF has demonstrated great potential for the early propagation of valuable animals, in particular, transgenic animals.

Production of cloned goats after nuclear transfer using adult somatic cells.

The developmental potential of adult somatic nuclei after nuclear transfer (NT) into enucleated, in vitro-matured oocytes was evaluated in a dwarf breed of goat (BELE: Breed Early Lactate Early). Somatic donor cells were obtained from two different sources: 1) adult granulosa cells (GCs) and 2) fetal fibroblasts. Primary GCs were obtained from follicular aspirants after laparoscopic oocyte pick-up (LOPU) and were cryopreserved immediately. Frozen aliquots of cells were thawed and cultured until confluent and were then cultured in low serum for 4 days before use in NT. Immature oocytes were obtained by LOPU and matured before enucleation and NT. Ninety-one adult GC-derived NT embryos were transferred into eight recipients, four of which were confirmed pregnant (50%) at Day 30 by ultrasound. Fifty-four male fetal fibroblast-derived NT embryos were transferred into six recipients, one of which was confirmed pregnant (17%). All pregnancies were maintained through term. Four recipients delivered seven female kids (three sets of twins) derived from the GC cultures (7.7% of embryos transferred). The other recipient delivered two male kids (3.7% of embryos transferred). Birth weights were within the normal range for dwarf goats. One female twin and one male twin died at birth; the remaining kids appeared healthy and normal. DNA analysis confirmed that the kids were genetically identical to their respective donors. These results demonstrated that adult caprine somatic cells could direct normal development after NT.

Expression of a reporter gene after microinjection of mammalian artificial chromosomes into pronuclei of bovine zygotes.

The introduction of mammalian artificial chromosomes (ACs) into zygotes represents an alternative, more predictive technology for the production of recombinant proteins in transgenic animals. The aim of these experiments was to examine the effects of artificial chromosome microinjection into bovine pronuclei on embryo development and reporter gene expression. Bovine oocytes aspirated from 2-5 mm size follicles were matured in vitro for 22 hr. Mature oocytes were fertilized in vitro with frozen- thawed bull spermatozoa. Artificial chromosome carrying either beta-galactosidase (Lac-Z) gene or green fluorescence protein (GFP) gene were isolated by flow cytometry. A single chromosome was microinjected into one of the two pronuclei of bovine zygotes. Sham injected zygotes served as controls. Injected zygotes were cultured in G 1.2 medium for 7 days. Hatched blastocysts were cultured on blocked STO cell feeder layer for attachment and outgrowth of ICM and trophectoderm cells. The results showed a high zygote survival rate following LacZ-ACs microinjection (74%). However, the blastocyst development rate after 7 days of culture was significantly lower than that of sham injected zygotes (7.5 vs. 22%). Embryonic cells positive for Lac-Z gene were detected by PCR in three of nine outgrowth colonies. In addition, GFP gene expression was observed in 15 out of 85 (18%) embryos at the arrested 2-cell stage to blastocyst stage. Six blastocysts successfully outgrew, three outgrowths were GFP positive for up to 3 weeks in culture. We conclude that the methodology for artificial chromosome delivery into bovine zygotes could lead to viable blastocyst development, and reporter gene expression could be sustained during pre-implantation development.

Overexpressing eukaryotic translation initiation factor 4E stimulates bovine mammary epithelial cell proliferation.

BACKGROUND AND AIMS: The eukaryotic translation initiation factor 4E regulates the proliferation of many cell types. In the present study, the effect of its overexpression on the growth of an immortalized bovine mammary epithelial cell line, MAC-T, has been investigated. Since involvement of cyclin D1 in growth regulation of other cell types has been suggested previously, the differences in cyclin D1 expression among the 4E-overexpressing and parental cells were also investigated. METHODS: The cDNA of mouse eukaryotic translation initiation factor 4E coding region (either wild-type or mutant, where Trp-56 was mutated to Ala) was transfected into MAC-T cells, and its protein expression was detected by Western blot analysis. Growth rates and saturation densities were calculated based on the cell counting data at desired time points. KEY RESULTS: The cells overexpressing wild-type 4E displayed higher growth rates and saturation densities compared to the parental cells (P<0.05), whereas cells expressing mutant 4E showed lower growth rates and saturation densities than the parental controls (P<0.05). The amounts of cyclin D1 mRNA and protein were higher in the wild-type transfectants than in the parental controls, whereas the mutant transfectants contained lower amounts of cyclin D1 mRNA and protein compared to the parental cells. CONCLUSION: Our results suggest that overexpression of eukaryotic translation initiation factor 4E leads to increased cyclin D1 expression at the transcriptional level, which consequently stimulates the proliferation of MAC-T cells.

Generation of dwarf goat (Capra hircus) clones following nuclear transfer with transfected and nontransfected fetal fibroblasts and in vitro-matured oocytes.

The developmental potential of caprine fetal fibroblast nuclei after in vitro transfection and nuclear transfer (NT) into enucleated, in vitro-matured oocytes was evaluated. Fetal fibroblasts were isolated from Day 27 to Day 30 fetuses from a dwarf breed of goat (BELE: breed early lactate early). Cells were transfected with constructs containing the enhanced green fluorescent protein (eGFP) and neomycin resistance genes and were selected with G418. Three eGFP lines and one nontransfected line were used as donor cells in NT. Donor cells were cultured in Dulbecco minimum Eagle medium plus 0.5% fetal calf serum for 4-8 days prior to use in NT. Immature oocytes were recovered by laparoscopic ovum pick-up and matured for 24 h prior to enucleation and NT. Reconstructed embryos were transferred as cleaved embryos into synchronized recipients. A total of 27 embryos derived from transgenic cells and 70 embryos derived from nontransgenic cells were transferred into 13 recipients. Five recipients (38%) were confirmed pregnant at Day 35 by ultrasound. Of these, four recipients delivered five male kids (7.1% of embryos transferred) derived from the nontransfected line. One recipient delivered a female kid derived from an eGFP line (7.7% of embryos transferred for that cell line). Presence of the eGFP transgene was confirmed by polymerase chain reaction, Southern blotting, and fluorescent in situ hybridization analyses. Nuclear transfer derivation from the donor cells was confirmed by single-strand confirmation polymorphism analysis. These results demonstrate that both in vitro-transfected and nontransfected caprine fetal fibroblasts can direct full-term development following NT.

Production of recombinant human type I procollagen homotrimer in the mammary gland of transgenic mice.

The large scale production of recombinant collagen for use in biomaterials requires an efficient expression system capable of processing a large (> 400 Kd) multisubunit protein requiring post-translational modifications. To investigate whether the mammary gland of transgenic animals fulfills these requirements, transgenic mice were generated containing the alpha S1-casein mammary gland-specific promoter operatively linked to 37 Kb of the human alpha 1(I) procollagen structural gene and 3' flanking region. The frequency of transgenic lines established was 12%. High levels of soluble triple helical homotrimeric [(alpha 1)3] type I procollagen were detected (up to 8 mg/ml) exclusively in the milk of six out of 9 lines of lactating transgenic mice. The transgene-derived human procollagen chains underwent efficient assembly into a triple helical structure. Although proline or lysine hydroxylation has never been described for any milk protein, procollagen was detected with these post-translational modifications. The procollagen was stable in milk; minimal degradation was observed. These results show that the mammary gland is capable of expressing a large procollagen gene construct, efficiently assembling the individual polypeptide chains into a stable triple helix, and secreting the intact molecule into the milk.

Toward altering milk composition by genetic manipulation: current status and challenges.

The implementation of large-scale genome mapping and sequencing has improved the understanding of animal genetics. A large number of gene sequences are now available to serve as regulatory elements or genes of interest. Although the central thrust of this work is focused on understanding disease states, the manipulation of normal metabolic processes is feasible. To date, the genetic manipulation of livestock has been limited to the permanent addition of genes of clinical interest. This study explores the utility of genetically engineered cattle as a means of altering milk composition to improve the functional properties of milk, increasing marketability. Improvements would include increasing the concentration of valuable components in milk (e.g., casein), removing undesirable components (e.g., lactose), or altering composition to resemble that of human milk as a means of improving human neonatal nutrition. The protracted time lines of genetically modifying dairy cattle has prompted the development of animal models. A model for dwarf goats is discussed in terms of circumventing the lengthy time lines involved in generating transgenic cattle and allowing for an accelerated expansion of research in molecular genetics of dairy animals. Thus, the genetic manipulation of dairy cattle is feasible and could have significant impacts on milk quality, attributes of novel dairy products, and human health.

Changes in expression of the prolactin and growth hormone gene during different reproductive stages in the pituitary gland of turkeys.

The changes in levels of prolactin (PRL) and growth hormone (GH) in plasma and the pituitary gland and their transcripts were measured in turkey hens at different physiological stages by radioimmunoassay and dot blot hybridization analysis, respectively. The levels of tPRL mRNA in the pituitary gland increased from those of the immature group to the egg-laying group, reaching a maximum during the incubation and a minimum during the moulting stages. Changes in pituitary levels of PRL and PRL mRNA followed a similar trend and consequently were highly correlated (r2 = 0.83), whereas a significant but lower correlation was observed between circulating and pituitary levels (r2 = 0.62). Less significant changes were measured for tGH mRNA, with maximum and minimum levels measured in the pituitaries of egg-laying and non-laying hens, respectively. These data suggest that although changes in concentration of PRL are correlated with the reproductive stage of the turkey hen, coordinate changes in levels of GH are not.

Mammary gland-specific hypomethylation of Hpa II sites flanking the bovine alpha S1-casein gene.

In the lactating cow, mammary gland-specific hypomethylation occurs at two Hpa II sites in the 5'-flanking region of the alpha S1-casein gene, and one in the 3'-region. These sites, A, B and C, are at nucleotide position -1388, -774 and +18034, respectively, relative to the major transcription start site. Site B was hypomethylated when the alpha S1-casein gene was expressed, and methylated when not expressed. In transgenic mice containing the bovine alpha S1-casein 5' and 3' regulatory elements fused to the human lactoferrin (hLF) cDNA, in some cases similar methylation patterns of sites A and B, as compared to the situation in the cow, were observed. In five mouse lines (out of the seven analysed) expressing the transgene in the milk, site B was hypomethylated in the mammary gland, while it was methylated in liver. In the two other mouse lines, no correlation was found between transgene expression and mammary gland-specific hypomethylation of site B. One of the five mouse lines with transgene expression and showing mammary-gland-specific hypomethylation of site B was studied in detail. In this mouse line, induction of transgene expression preceded hypomethylation of site B.

Development and validation of a homologous radioimmunoassay using a biologically active recombinant turkey prolactin.

1. A new homologous radioimmunoassay has been developed for the measurement of turkey prolactin. 2. A 25000 kDa purified recombinant derived turkey prolactin (rtPRL), the biological activity of which was tested using a crop sac assay, was used as immunogen for the production of rabbit antiserum. In this biological test, the rtPRL was as active as the ovinePRL. 3. The radioligand (rtPRL) was labelled with 125I and the assay allowed the detection of standard doses of rtPRL ranging from 400 pg/tube to 50 ng/tube. 4. No cross reaction with chicken luteinising hormone and recombinant chicken growth hormone was detected. 5. The within and between assay coefficients of variability were 5.0 +/- 2.7% and 16.3%, respectively. The overall mean recovery ratio was 1.01. 6. The dose-response curves obtained with serial dilution of plasma and pituitary from turkey hens at different physiological stages and from male turkeys were parallel to those obtained with standard rtPRL. 7. The measured concentration of prolactin was 5 times higher in plasma from incubating than laying turkey hens, and the pituitaries from incubating hens contained 2 and 4 times more prolactin than those of laying and out of lay hens or males, respectively. 8. To further assess the validity of the assay, we measured changes in plasma concentration of prolactin in turkeys following stimulation with chicken vasointestinal peptide (cVIP). A single injection of 1 or 10 micrograms/kg body weight of cVIP to laying hens produced a large and rapid increase in plasma prolactin. 9. This new radioimmunoassay appears to be high for the measurement of turkey prolactin.

Production and characterization of recombinant turkey prolactin.

1. Recombinant turkey prolactin (rctPRL) was produced as a fusion protein in E. coli, purified by affinity chromatography followed by cleavage with thrombin. The final yield of the released rctPRL (> 90% purity) was 1-2 mg/l of bacterial culture. 2. Recombinant tPRL co-migrated with the main immunoreactive band (25 kDa) in turkey pituitary extracts and was identical to natural tPRL except for the addition of three amino acids (Gly-Ser-Ser) resulting from the cloning strategy at the amino terminal end. 3. The bioactivity of the rctPRL was equipotent to ovine PRL in a rabbit mammary explant system and in the Nb2 lymphoma mitogenic assay.