Harry M. Meade

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.

Transgenic milk as a method for the production of recombinant antibodies

Abstract Recombinant antibodies and their derivatives are increasingly being used as therapeutic agents. Clinical applications of antibodies often require large amounts of highly purified molecules, sometimes for multiple treatments. The development of very efficient expression systems is essential to the full exploitation of the antibody technology. Production of recombinant protein in the milk of transgenic dairy animals is currently being tested as an alternative to plasma fractionation for the manufacture of a number of blood factors (human antithrombin, human alpha-1-antitrypsin, human serum albumin, factor IX). The ability to routinely yield mg/ml levels of antibodies and the scale-up flexibility make transgenic production an attractive alternative to mammalian cell culture as a source of large quantities of biotherapeutics. The following review examines the potential of transgenic expression for the production of recombinant therapeutic antibodies.

14 – EXPRESSION OF RECOMBINANT PROTEINS IN THE MILK OF TRANSGENIC ANIMALS

Over 50 proteins have been expressed in the milk of transgenic animals. This chapter focuses on the status of expression of recombinant proteins in such milk. The chapter discusses the expression of therapeutically useful proteins in mammalian milk, the emphasis being on dairy animals as the production systems of choice. The chapter presents an overview of the expression constructs, milk-specific transgenes, transgene insertion, transgenic animal production, milk, and protein purification. The quality regulatory issues related to this are also discussed. The chapter also states that the mammary bioreactor is capable of most posttranslational modifications and protein folding, and can be used to produce complex proteins. The chapter presents the current status where transgenic dairy animals are providing a bulk protein production system. This system is capable of making proteins available that are either not available today or are only recoverable from other sources. Transgenic production may thus enable a move from “on-demand” patient treatment to prophylaxis and far wider indications and use of many proteins. In addition, the production of proteins in the milk of transgenic dairy animals is highly cost-effective, opening up real possibilities for nutraceutical product development.

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.

Antitransferrin receptor antibody-RNase fusion protein expressed in the mammary gland of transgenic mice.

Antibodies fused to human enzymes offer an alternative to specifically targeting tumors with antibodies linked to plant or bacterial toxins. Since large amounts of these reagents can be administered without eliciting non-specific toxicities, efficient methods of production are needed. The goal of this work was to express a complex immunoenzyme fusion protein (immunotoxin) in the mammary gland of transgenic mice. A chimeric mouse/human antibody directed against the human transferrin receptor (E6) was fused at its CH2 domain to the gene for a human angiogenic ribonuclease, angiogenin (Ang). It was expressed in the mammary gland of mice and secreted into mouse milk. Expression levels in milk were approximately 0.8 g/l. The chimeric protein retained antibody binding activity and protein synthesis inhibitory activity equivalent to that of free Ang. It was specifically cytotoxic to human tumor cells in vitro.

Expression of a Bovine κ-CN cDNA in the Mammary Gland of Transgenic Mice Utilizing a Genomic Milk Protein Gene as an Expression Cassette

Transgenic mice were produced by microinjection of a DNA construct composed of the bovine κ-casein (κ-CN) cDNA under the control of the goat β-CN 5′ promoter elements and 3′ flanking regions into pronuclear-stage embryos. The gene construct targeted the expression of bovine κ-CN RNA to the mammary gland and secretion of bovine κ-CN in the milk. In the three lines studied (BC-7, BC-31 and BC-67) the transgene was stably integrated and propagated as a Mendelian locus. Expression of the bovine protein in lactating mice from the three transgenic lines was demonstrated by northern and western blots. In ten different tissues analysed by northern blotting, expression was confined to the mammary gland of lactating transgenic mice from line BC-7, with low-level expression also observed in the salivary gland of lines BC-31 and BC-67. Transgene expression in the mammary gland paralleled normal casein gene expression during lactation and was not observed in virgin females. The level of bovine κ-CN mRNA expression on day 10 of lactation in hemizygous transgenic females in relation to endogenous mRNA of whey acid protein (WAP) gene expression was 14%, 69% and 127% in lines BC-7, BC-31 and BC-67, respectively. No association between transgene copy number and expression was observed. The bovine κ-CN concentration in milk on day 10 of lactation ranged from 0.94 to 3.85 mg of protein per ml of milk. The bovine κ-CN expressed in mouse milk had the same molecular mass and immunoactivity with polyclonal antibodies as did κ-CN from bovine milk. A high degree of variation in the production of bovine κ-CN within each of the transgenic lines was observed.

Toward a new cash cow

Cloned cattle engineered to carry an artificial chromosome encoding human immunoglobulin genes are a significant leap toward the production of safer and more potent therapeutic antibodies.

Neutralization of HIV by milk expressed antibody.

Background:In some areas of the world, mother-to-child transmission of HIV remains a significant problem in part due to widespread breastfeeding, which is essential because of scarce supply of a safe replacement, protection conferred by breast milk against many enteric illnesses, and cultural norms. We propose that sustained adequate levels of protective antibodies in breast milk will prevent transmission of HIV. Methods:The HIV-neutralizing human monoclonal antibody b12 (IgG1) has been expressed as an IgA2 in CHO cells and shown to retain full immunoreactivity and neutralizing activity as the parental IgG1. The expression plasmids containing the b12 heavy and light chains were also used to construct milk-specific expression vectors using the GTC goat &bgr;-casein expression vector to direct expression of linked genes to the mammary gland with subsequent secretion into the milk. Female transgenic mice were generated and following parturition, their milk was tested for antibody immunoreactivity with gp120 and neutralization of HIV. Results:When milk-derived b12 IgA2 was compared with CHO-derived b12 IgA2 (or IgG1), immunoreactivity was retained. When tested for neutralization, milk-derived b12 IgA2 was at least comparable to CHO-derived antibody and in some cases, superior to CHO-derived antibody. Furthermore, milk that expressed b12 IgA2 was significantly more effective at mediating antibody-dependent cell killing. Conclusions:These results suggest that it is possible to achieve functional HIV-specific mAb in the milk of transgenic mice, and further investigations are warranted to explore ways for inducing this type of antibody response in the breast milk of HIV-infected women.

Transgenic Cloned Goats and the Production of Recombinant Therapeutic Proteins

The clinical use of recombinant therapeutic proteins has increased significantly during the last few decades. However, therapeutic indications often require large amounts of highly purified product, especially for therapies that call for chronic dosing regimes. The development of very efficient expression systems is essential to the full exploitation of the recombinant technology, so that life-saving medicines will become more readily available. Transgenic production technology is a highly scalable, capital-sparing approach to the manufacturing of complex recombinant proteins. ATryn®, recombinant antithrombin purified from the milk of transgenic goats, the first transgenically-derived therapeutic protein to gain regulatory approval, is currently approved in the United States and the EU for the treatment of Hereditary Antithrombin Deficiency. In addition, production of additional recombinant proteins in the milk of transgenic goats and rabbits is currently being tested for the production of a number of therapeutic antibodies as well as an alternative to plasma fractionation for the manufacture of human plasma proteins. The following review examines the potential of somatic cell nuclear transfer to generate transgenic goats used in the production of human recombinant therapeutics.

Transgenic Cloned Goats and the Production of Therapeutic Proteins

Publisher Summary The use of recombinant therapeutic proteins has increased during the past two decades. Clinical applications often require large amounts of highly purified molecules, sometimes for multiple treatments. The development of very efficient expression systems is essential for the full exploitation of recombinant technology. Production of recombinant protein in the milk of transgenic goats is currently being tested for the production of a number of therapeutic antibodies as well as an alternative to plasma fractionation for the manufacture of human antithrombin. This chapter focuses on the potential of somatic cell nuclear transfer to generate transgenic goats used in the production of recombinant therapeutics. The nuclear transfer protocols offer several advantages over the use of pronuclear microinjection such as preselection of transgenic integration sites, nonmosaic founder, and apparent reduction in the number of surgical procedures necessary to generate founders. The goats generated by somatic cell nuclear transfer do not suffer from health problems such as large birth weight, placentation defects, and hematological dysfunction, which are sometimes reported with cloned sheep and cattle.