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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., (Copyright © 2011 Wiley-Liss, Inc.)

Relaxin is a peptide hormone produced by a wide variety of mammals. In the horse, the placenta is the major source of relaxin. Since pure equine relaxin is difficult to obtain to study its role in the pregnant mare, the objectives of this study were to produce recombinant equine prorelaxin and characterize its immunological and biological activity. First, an equine relaxin gene cassette was transfected into immortalized bovine mammary epithelial (MAC-T) cells. Second, immunological activity of media conditioned by transfected MAC-T cells was tested by Western blotting and quantified using a homologous equine radioimmunoassay. Finally, bioactivity of the conditioned media was tested using the human monocyte cell line, THP-1, which exhibits a rapid and dose-dependent increase in the accumulation of cAMP upon binding relaxin. The results showed that conditioned media, concentrated 5x, yielded 4.11 +/- 0.81 ng/ml recombinant equine prorelaxin. In addition, a 19 kDa immunoreactive band, corresponding to the expected size of equine prorelaxin, was visualized by SDS-PAGE. THP-1 cells incubated with conditioned media (5x) from transfected cells, in the presence of forskolin (1 microM) and isobutylmethylxanthine (50 microM), showed an increase in cAMP production over media from mock-transfected cells alone. In conclusion, recombinant equine prorelaxin secreted by MAC-T cells was both immunologically and biologically active. This study demonstrates the first attempt to produce recombinant equine prorelaxin, important for further study of the role of relaxin in the mare.

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.

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.

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.

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.

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., (Copyright 2002 Wiley-Liss, Inc.)

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.

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., (Copyright 2001 Wiley-Liss, Inc.)

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.

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.

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.

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.

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.

Objectives: The possible role of sex hormone imbalance in hepatocellular carcinogenesis was investigated., Patients and Methods: Ten men with hepatocellular carcinoma (HCC) and cirrhosis, 10 men with HCC without cirrhosis, 12 men with cirrhosis of various aetiologies, 8 men with secondary liver tumours and 10 normal men were studied. Plasma levels of testosterone, androstenedione, oestradiol, sex hormone binding globulin follicle stimulating hormone and luteinizing hormone were determined for all patients., Results: Patients of all groups had comparable levels of oestradiol, androstenedione, sex hormone binding globulin, luteinizing hormone and follicle stimulating hormone. Low testosterone values in the serum were found for all patients with primary liver disease. Low testosterone was found even in patients with primary HCC who did not have cirrhosis or liver failure serious enough as to be responsible for the reduction of testosterone levels. On the other hand patients suffering from secondary liver tumours had normal values of serum testosterone., Conclusions: These results present an indication that low serum testosterone is a special feature of hepatocellular carcinoma.

A rapid and sensitive chromatographic method is described for determining desmosine, isodesmosine, 5-hydroxylysine, tryptophan, lysinoalanine, glucosamine and galactosamine at picomole levels in protein and tissue hydrolysates. This method uses either an automated amino acid analyser with a 17.5 X 0.28 cm microcolumn packed with 6.0 +/- 0.5 micron spherical resin, thermostated at 52 degrees C, one buffer system (0.21 M sodium citrate, pH 5.125) and 3-nitrotyrosine as the internal standard, or conventional instruments using the same system but with larger diameter columns and resins (11.0 +/- 1.0 micron). This method should be especially valuable for determining collagen and elastin in tissue hydrolysates from the amounts of 5-hydroxylysine, and desmosine or isodesmosine present, respectively, and for studying protein hydroxylation, glycosylation, cross-linking formation, and the turnover rates of collagen and elastin in normal and diseased tissues.

Intracellular and extracellular skeletal muscle protein fractions were isolated from the legs and breasts of young and adult White Leghorn chickens and quantified by detailed amino acid analysis. This involved repeated homogenization in the presence of 50 mM CaCl2, neutral phosphate-buffered saline (pH 7.4), solubilization by 2% sodium dodecyl sulfate (SDS), and centrifugation to separate all intracellular muscle proteins from the extracellular matrix. The total SDS-soluble intracellular muscle proteins in the adult and young birds ranged respectively from 93.2 to 94.5% in the leg and from 93.5 to 94.1% in the breast muscles. Collagen and collagen-like proteins in the extracellular matrix protein fractions were calculated from the amounts of 5-hydroxylysine found in their 96-h acid hydrolysates and elastin content from the amounts of desmosine present. Total collagen ranged from 3.42 to 5.18% in legs and from 2.91 to 3.89% in breasts. The elastin content of leg muscles represents only .061% of the total muscle protein. The calculated protein efficiency ratios for intracellular avian muscle proteins averaged 3.2 compared with a mean value of 1.4 for the extracellular matrix.

Analytical single-column chromatographic methods have been developed for determining all methylated basic amino acids, isodesmosine, desmosine, the amino sugars glucosamine and galactosamine, the diastereoisomers of 5-hydroxylysine, and related compounds at picomole levels in protein and tissue hydrolysates. Complete resolution of all these unique basic amino acids as discrete peaks was achieved in 5.4 on a 50 X 0.28-cm microcolumn of Dionex type DC-4A spherical resin (9.0 +/- 0.5 micron) using updated instrumentation commonly available for amino acid analysis. The column was operated at 5.65 ml/h with two 0.35 M sodium citrate buffers (pH 5.700 and 4.501), at two temperatures (31.5 and 73 degrees C). Excellent resolution of all omega-N-methylarginines and related compounds was also achieved in 3 h using a 17.5 X 0.28-cm microcolumn of Dionex DC-5A resin (sized to 6.0 +/- 0.5 microns), two citrate buffers (0.21 M Na+, pH 5.125; 0.35 M Na+, pH 5.700), a buffer flow rate of 5.75 ml/h, and a temperature of 52 degrees C. Complete separation of all other amino acids found in protein or tissue hydrolysates including S-carboxymethyl cysteine, 4-hydroxyproline, methionine S,S-dioxide, and the amino sugars was also carried out in 95 min using a 23.5 X 0.28-cm microcolumn of Dionex DC-5A resin. The use of purified microcolumn buffers gave smooth baselines without interference from artifacts or minor hydrolysate components. The major advantages of these methods are: first, their high resolving power; second, their high sensitivity which is comparable and in some aspects superior to the newer instruments; and third, their high reproducibility (100 +/- 2.5%) and low operating costs. These methods should be especially valuable for determining myosin, actin, and elastin in tissue hydrolysates from the amounts of N tau-methylhistidine, desmosine, or isodesmosine present, respectively, and for studying protein methylation, hydroxylation, cross-linking formation, and the turnover rates of contractile and connective tissue proteins in biological systems.