Recombinant antithrombin: production and role in cardiovascular disorder.

Plasma-derived antithrombin (AT) concentrates have been used for the management of hereditary and acquired deficiencies since the early 1980s. Recombinant versions of other blood factors and their derivatives are increasingly becoming available, providing a safe and abundant supply of these important therapeutics. However, the complexity of the AT molecule and the large doses often required for supplementation treatments preclude the use of traditional cell culture bioreactors for recombinant production. The development of a very efficient expression system has been necessary for the cost-efficient recombinant production of AT. Transgenic production, with its ability to yield high levels of heterologous protein and its scale-up flexibility, is an attractive alternative to plasma fractionation. Purification of recombinant AT from the milk of transgenic dairy goats has been developed to provide a homogeneous, well-defined, and abundant supply of this factor. This article describes the production of recombinant AT and aspects of clinical applications of this molecule to cardiovascular disorders.

Transgenic production from in vivo-derived embryos: effect on calf birth weight and sex ratio.

We examined transgenic-cattle production by DNA microinjection into 1-, 2-, and 4-cell embryos, analyzing the impact on calf size and subsequent viability. Embryos were either collected at an abattoir by flushing oviducts from superovulated and artificially inseminated cows (in vivo-derived) or obtained by in vitro maturation and in vitro fertilization of oocytes aspirated from excised ovaries (in vitro-derived). A human serum albumin (hSA) milk-expression DNA construct was microinjected, either in one of the visible pronuclei of in vitro- and in vivo-derived 1-cell embryos or in the nuclei of two blastomeres of 2- and 4-cell in vivo-derived embryos. Microinjection-induced mortality (lysis and developmental block) was equivalent ( approximately 40%) for all microinjected embryos. Embryos were co-cultured with BRL cells in B-2 medium containing 10% fetal calf serum (FSC). Overall, embryo development to morulae/blastocysts was significantly greater for in vivo-derived ova (15.5%) than for in vitro-derived oocytes (9.3%). All morulae and blastocysts were transferred to synchronized recipient females on Days 6-8 post-fertilization. A total of 189 calves were delivered. Birth weights were significantly greater for calves generated from in vitro-derived oocytes compared with those generated from in vivo-derived oocytes. One transgenic bull calf was obtained from the microinjection of a 2-cell embryo. Fluorescence in situ hybridization (FISH) analysis of lymphocytes detected one transgenic integration site in all cells. Transmission frequency of the hSA transgene in embryos obtained through IVM/IVF/IVC utilizing the semen of the transgenic calf confirmed that it was not mosaic.

Hormonal induced lactation in transgenic goats.

The aim of this study was to hormonally induce lactation in prepubertal, nulliparous, and male goats both transgenic and non-transgenic. Analysis of milk quality, recombinant protein expression levels, total amount of recombinant protein produced, and the affect on long-term reproductive capability was assessed. Fifty-one goats (Saanen, Alpine, and Toggenburg), male and non-pregnant females, 2-31 months of age, either non-transgenic or transgenic were evaluated with a total of 10 transgenes (constructs) represented. Animals were given estradiol (0.25 mg/kg, i.m.) and progesterone (0.75 mg/kg, i.m.) on days 1, 3, 5, 7, 9, 11 and 13, while prednisilone (0.4 mg/kg, i.m.) was administered on days 14-16 with mammary massage occurring daily from day 5 onward. Forty of 51 animals, (36 of 38 females and 4 of 13 males) produced milk with total volumes in the 30-day experiment, ranging from 20 microl to 530 mls per day, or approximately 500 microl to 6.8 liters total. Milk composition was analyzed for various parameters (total protein, fat content, total solids and somatic cell count) with no significant differences found between induced and natural milk. Expression levels of recombinant proteins from transgenic animals that were analyzed during the induced lactation, and subsequently during normal lactations, were found to have no significant differences. Total amount of recombinant protein produced was evaluated at different expression levels with no statistical significance seen. While over 90% of the females placed in the regimen became pregnant, there was a correlation between increased age at time of induction and an increase in number of breedings, or reproductive cycles needed to establish a pregnancy after induction. For males, 100% placed in the regimen settled females after hormonal induction of lactation. Semen quality was evaluated prior to, during, and after hormonal treatments. Semen volume and sperm number did not differ; however, for a small percentage of males, there was a decrease in sperm and post thaw motility after hormonal treatments. These levels returned to normal within 4-5 weeks. Subsequent natural lactations showed total milk volumes within breed standards. These findings indicate that hormonal induction of lactation in the caprine species is a viable alternative to pregnancy for initiating lactation and milk production, does not adversely impact reproductive performance long-term, and can benefit the early assessment of recombinant proteins produced in a transgenic founder program.

Comparing follicle stimulating hormone from two commercial sources for oocyte production from out-of-season dairy goats.

Until recently, two sources of follicle stimulating hormone (FSH-P; Schering-Plough; Kenilworth, NJ and Super-Ov; FSH-SOV; AUSA International, Tyler, TX) have been commercially available in the United States and routinely used for superovulation of ruminants. Because there have been no comparative follicle stimulating hormone studies on small ruminants, we determined the difference between the number of follicles induced and the number of oocytes that can subsequently be harvested from goats stimulated with either of these two follicle stimulating hormone products. Anestrous Saanen does were fitted with a progestin implant then randomly assigned to one of two ovarian stimulation groups. Starting 4 d after introducing the progestin implant, donors in treatment 1 were administered daily injections of FSH-P for 4 d. Does in treatment 2 were similarly treated but were administered FSH-SOV for 4 d. Follicle aspirations were performed by laparotomy in the morning of treatment d 8. In summary, no difference was detected between the two stimulatory agents for the number of follicles and quality of oocytes harvested from stimulated does, indicating that these two commercial FSH products could be used successfully for ovarian stimulation of anestrous dairy goats.

Production of goats by somatic cell nuclear transfer.

In this study, we demonstrate the production of transgenic goats by nuclear transfer of fetal somatic cells. Donor karyoplasts were obtained from a primary fetal somatic cell line derived from a 40-day transgenic female fetus produced by artificial insemination of a nontransgenic adult female with semen from a transgenic male. Live offspring were produced with two nuclear transfer procedures. In one protocol, oocytes at the arrested metaphase II stage were enucleated, electrofused with donor somatic cells, and simultaneously activated. In the second protocol, activated in vivo oocytes were enucleated at the telophase II stage, electrofused with donor somatic cells, and simultaneously activated a second time to induce genome reactivation. Three healthy identical female offspring were born. Genotypic analyses confirmed that all cloned offspring were derived from the donor cell line. Analysis of the milk of one of the transgenic cloned animals showed high-level production of human antithrombin III, similar to the parental transgenic line.

Transvaginal ultrasound-guided oocyte retrieval following FSH stimulation of domestic goats.

The objectives of this study were to evaluate different ovarian stimulation protocols on donor goats and to develop a safe, repeatable method for harvesting oocytes from FSH-treated does (Experiment I). Based on the preliminary findings of the first experiment, 32 crossbred does were used in a second experiment (Experiment II), 16 that had not been previously aspirated and 16 that had undergone one previous aspiration, were used to fine tune the procedure. Females were randomly subjected to 1 of the 2 ovarian stimulation protocols: Treatment (A) does were implanted with a norgestomet ear implant. Starting 10 d post-implantation, does were administered FSH daily for 4 d. Does in Treatment (B) were treated similarly to those in (A) but were implanted for only 3 d before starting the FSH injections and implants were not removed prior to aspiration. Using a 2 x 2 factorial arrangement, fresh does (n=16), not previously aspirated, were then further randomly assigned to either a laparoscopic aspiration procedure (LAP) or a transvaginal ultrasound-guided aspiration procedure (TUGA). The LAP procedure was performed using a fiber optics. For the TUGA, the doe was placed in dorsal recumbency, and a 5 MHz human transvaginal transducer, attached to the ultrasound unit, was positioned vaginally for oocyte aspiration. In summary, there was no significant difference among treatment groups for parameters evaluated, with the exception of methods for oocyte collection. The number of follicles detected and oocytes harvested using TUGA (9.5 and 4.3, respectively) was less than for females obtained by LAP (17.4 and 14.4, respectfully). The percentage of oocytes recovered from does subjected to the TUGA (68%), however, was similar to those subjected to the LAP (69%). Unlike donor does subjected to a repeated LAP, there was no evidence of adhesions in donor does from the repeated TUGA group. The TUGA approach to oocyte collection should not be overlooked in an effort to decrease the chances of adhesions in valuable donor goats.

Transgenically produced human antithrombin: structural and functional comparison to human plasma-derived antithrombin.

Recombinant human antithrombin (rhAT) produced in transgenic goat milk was purified to greater than 99%. The specific activity of the rhAT was identical to human plasma-derived AT (phAT) in an in vitro thrombin inhibition assay. However, rhAT had a fourfold higher affinity for heparin than phAT. The rhAT was analyzed and compared with phAT by reverse phase high-performance liquid chromatography, circular dichroism, fluorophore-assisted carbohydrate electrophoresis (FACE), amino acid sequence, and liquid chromatography/mass spectrography peptide mapping. Based on these analyses, rhAT was determined to be structurally identical to phAT except for differences in glycosylation. Oligomannose structures were found on the Asn 155 site of the transgenic protein, whereas only complex structures were observed on the plasma protein. RhAT contained a GalNAc for galactose substitution on some N-linked oligosaccharides, as well as a high degree of fucosylation. RhAT was less sialylated than phAT and contained both N-acetylneuraminic and N-glycolylneuraminic acid. We postulate that the increase in affinity for heparin found with rhAT resulted from the presence of oligomannose-type structures on the Asn 155 glycosylation site and differences in sialylation.

Commercialization of proteins produced in the mammary gland.

In the mid 1980's, few pioneering companies undertook the risk of developing methodologies for the production of complex human therapeutic proteins in the milk of transgenic animals. As we approach the end of the 1990's, the prospect of achieving this aim is becoming a reality as the first of these human therapeutic products, antithrombin III and alpha-1-antitrypsin are making their way through human clinical trials. It is projected that licensure by the Regulatory agencies and market launch for these transgenically produced therapeutics will occur around the year 2000. Although much has already been achieved, additional transgenic challenges await the basic embryo researcher and practitioner. The biopharming community recognizes the need for additional innovative methodologies (such as cloning, sperm sexing and retroviral mediated gene transfer etc.) to overcome the natural biological barriers and increase the efficiency of transgenic dairy animal production and rapid herd expansion.

Analysis of glutaminase activity and RNA expression in preimplantation mouse embryos.

Glutamine is utilized as an energy substrate in preimplantation mouse embryos. Glutaminase is the enzyme responsible for the conversion of glutamine to glutamic acid, which then enters the trichloro acetic acid (TCA) cycle as alpha-ketoglutarate. Glutaminase enzyme activity was assessed in preimplantation embryos that developed in vivo, and glutaminase RNA expression was examined in embryos that developed in vivo or were cultured in CZB medium to various preimplantation stages between 1-cell and blastocyst. Glutaminase activity in 1-8-cell-stage mouse embryos that developed in vivo ranged from 0.009-0.01 U/mg protein (2.39-2.95 x 10(-7) U per embryo) and increased 3-4 fold to 0.034 U/mg protein (8.13 x 10(-7) U per embryo) at the blastocyst stage. Relative stage-specific expression of glutaminase RNA was assessed by reverse transcription polymerase chain reaction (RT-PCR) in embryos that developed both in vivo and in CZB culture. In vivo, glutaminase RNA was expressed at the 1-cell stage, declined to 23% of 1-cell levels at the early 2-cell stage, and reaccumulated from late 2-cell through blastocyst stage, where it reached a high of 204% of 1-cell levels. CZB-cultured embryos exhibited a similar pattern of developmental RNA expression, declining to 30% of 1-cell levels at the early 2-cell stage, and increasing RNA expression at the blastocyst stage to 191% of the 1-cell level.

Minimization of viral contamination in human pharmaceuticals produced in the milk of transgenic goats.

The minimization of viral contamination in therapeutic proteins produced in transgenic goats' milk can be achieved by a combinatorial approach. It begins with reduction in the risk in the starting material followed by appropriate clearance/inactivation steps in the purification process. To minimize risk in the starting material, Genzyme Transgenics Corporation (GTC)'s closed goat herds are subjected to routine serological surveillance for known viral diseases, especially those transmitted through milk. Although scrapie is defined as a slow-acting virus of sheep and goats, its incidence in goats in the US is rare (only four cases) and all four were in goats co-mingled with scrapie-infected sheep. All GTC's domestic goats were selected for previous non-exposure to sheep, cows or scrapie. In addition, milk, which is the starting material for transgenic protein production, is categorized as non-infectious for prions. Standard operating procedures are in place at GTC Farm sites to minimize human, animal or vehicular vectoring of viral diseases and the transgenic production animals are milked according to high standard Good Agricultural Practices (GAP). The transgenic protein (ATIII) purification process contains steps that should provide a high level of viral reduction. Validation of viral and prion removal will also be undertaken.

One-minute exposure of 4-cell mouse embryos to glucose overcomes morula block in CZB medium.

One-cell mouse embryos that block at the 2-cell stage can progress to the morula stage in CZB medium, but fail to cavitate and then swell and lyse. A 1-min exposure to 27 mM glucose at the 4-cell stage (approximately 42 hr) will support a high frequency of development to the blastocyst stage (75%) in the same medium. A glucose exposure is beneficial anytime between 30 and 54 hr of culture (67-73% blastocysts). Of a group of additional sugars and glucose analogues tested for their ability to replace glucose, only galactose was equivalent in promoting embryo development to the blastocyst stage (64% blastocysts).

Chemiluminescent detection of alkaline phosphatase in PhastGel.

A chemiluminescent assay has been applied to the detection of alkaline phosphatase on PhastGel containing lysates of preimplantation mouse embryos. The very sensitive detection capabilities reported for the chemiluminescent system led to the investigation of its applicability to the characterization of the alkaline phosphatases in one embryo or less and to compare the sensitivity of two different commercial alkaline phosphatase chemiluminescent assays to a colorimetric assay.

Embryonic alkaline phosphatase is expressed at M-phase in the spermatogenic lineage of the mouse.

We have recently cloned and characterized a novel embryonic alkaline phosphatase (EAP) expressed at the two-cell to blastocyst stage of preimplantation development in the mouse. The isozyme is re-expressed in trace amounts in the thymus, intestine and testis during adult life. In the present report, we find that EAP transcripts can be detected, by RT-PCR analysis, in very low amounts in the testes of newborn mice, but at 24 days of age EAP mRNA levels reach the highest concentrations, remaining high at 40 and even 117 days of age. We produced a synthetic peptide and a corresponding rabbit anti-peptide antiserum (Rb-1434), which was characterized by enzyme antigen immunoassays and reactivity with chinese hamster ovary cell transfectants, as reacting specifically with EAP. The Rb-1434 antibody enabled us to examine immunohistochemically what cell types in the testis are responsible for the expression of EAP during different developmental stages. No positive cells were recognized in the testis of newborns (day 0) and 8-day-old mice. Positive cells were first observed at day 15 and, at 24 days of age, many positive M-phase cells, morphologically corresponding to spermatocytes in mid to late prophase of meiotic division I, were strongly positive for EAP expression. Positive M-phase cells were also observed at 40 days and 151 days of age. Transgenic mice expressing the human GCAP isozyme in a tissue-specific manner in the testis, showed equivalent stages of M-phase figures when stained immunohistochemically with a specific rabbit polyclonal antiserum.(ABSTRACT TRUNCATED AT 250 WORDS)

Genomic structure and comparison of mouse tissue-specific alkaline phosphatase genes.

A full-length human placental alkaline phosphatase (AP) cDNA was used to identify and clone related genes from mouse genomic libraries. We report the cloning, sequence, and structural comparison of the mouse embryonic and intestinal AP genes and a putative AP pseudogene. All three mouse genes are composed of 11 exons interrupted by 10 small introns (70-261 bp) with an organization analogous to that of the three human tissue-specific AP genes. Introns interrupt the coding sequences at identical positions in all three mouse and human tissue-specific AP genes. The deduced amino acid sequence of the isozymes predicts proproteins of 529, 559, and 466 amino acids for embryonic AP, intestinal AP, and pseudo-AP, respectively. A repetitive sequence inserted in exon XI of the mouse intestinal AP gene codes for a unique stretch of 41 amino acids, 20 of which are threonines. This insertion has disrupted a region recognized as being responsible for phosphatidylinositol anchorage of human placental AP to the cytoplasmic membrane. Phylogenetic analysis indicates that the three mouse AP isozymes form a distinct group separate from the human tissue-specific AP isozymes, suggesting the taxon-specific evolution of the AP genes as opposed to independent evolution of AP genes expressed in specific tissues.

Differential regulation of G protein subunit expression in mouse oocytes, eggs, and early embryos.

Pertussis toxin ADP-ribosylation and Western blot analysis using G protein-specific antibodies were used to study G protein expression in mouse oocytes, eggs, and early embryos. A pertussis toxin (PT) substrate of about 40 kDa was observed in all stages, but its level was stage dependent. It decreased dramatically between germinal vesicle stage oocytes and unfertilized eggs, remained relatively constant through the early 2-cell stage, and then declined again with each cell division, reaching the lowest level at the 8- to 16-cell stage. Its level, or perhaps that of a different substrate, then increased at the blastocyst stage. Western blot analysis with antisera to the G protein alpha subunit indicated that the decrease between germinal vesicle stage oocytes and unfertilized eggs was less pronounced for the alpha subunit itself than for the PT substrate. Antisera to G protein beta subunit revealed that the difference in the amount of this subunit in germinal vesicle-stage oocytes versus unfertilized eggs was even greater than that of the PT ADP-ribosylation substrate. These results suggest that during oocyte maturation G protein beta gamma levels decline to a greater extent than alpha levels. Additional evidence supporting this hypothesis was obtained by showing that addition of exogenous beta gamma to unfertilized egg preparations increased the amount of PT substrate. These results indicate that G protein subunit expression is differentially regulated during oocyte maturation.

Polarization of blastomeres in the cleaving rabbit embryo.

Cellular polarization is believed to be a crucial event in the differentiative divergence of the two cell lineages leading to the blastocyst in rodent embryos. This study was undertaken to determine if rabbit embryos exhibited cellular polarization prior to blastocyst formation and to define the embryonic stage at which polarization was first apparent. Polarity was assayed by observation of the pattern of binding of FITC-Con A to dissociated blastomeres from three stages of rabbit embryos. Scanning electron microscopy on the dissociated cells confirmed the fluorescence results. Fifty-one percent of blastomeres in 38- to 66-cell rabbit embryos exhibited an intense pole of FITC-Con A binding and a single pole of microvilli. Only 2% of blastomeres at the 17- to 34-cell stage were similarly polarized and none were polarized at the 8- to 16-cell stage. In addition, during attempts to remove the mucin coat and zona pellucida from the rabbit embryos prior to their dissociation, it was found that the protease sensitivity of these coats also changed at the 38- to 66-cell stage. Prior to this time, although the mucin coat disappeared after 30 min in 0.5% pronase, the zona required approximately 1.5-2.5 hr in pronase for even partial removal. At the 38- to 66-cell stage, pronase dissolved the mucin coat within 10 min and the zona pellucida within 20 min. The zona was resistant to 0.1% proteinase K at all stages examined.

Two alkaline phosphatase genes are expressed during early development in the mouse embryo.

Alkaline phosphatase (AP) activity is stage specific in mouse embryos and may be associated with compaction and separation of trophectoderm from inner cell mass in preimplantation development. We previously sequenced a cDNA and two mouse AP genes that could contribute to the AP activity in embryos. Oligonucleotide primers were constructed from the three sequences and used in the reverse transcription-polymerase chain reaction technique to establish that two of the three AP isozymes are transcribed during preimplantation development. The predominant transcript (E-AP) is from a gene highly homologous to the human tissue-specific APs, but different from the mouse intestinal AP. Tissue non-specific (TN) AP also is transcribed, but there is approximately 10 times less TN-AP than E-AP transcript. The TN-AP isozyme is the predominant transcript of 7 to 14 day embryos and primordial germ cells. A switch in predominance from E-AP to TN-AP must occur during early postimplantation development. This study establishes a framework for experiments to determine the functions of the two isozymes during preimplantation development.