CHAPTER 2 1TRANSPHARMERS - BlOREACTORS FORPHARMACEUTICAL PRODUCTS The application of transgenic technology to commercially impor-tant livestock is expected to generate major effects in agriculture andmedicine. Three areas of development have been the focus of intensiveinvestigation: (1) For improved desirable traits, such as increased growthrate, feed conversion, reduction of fat, improved quality of meat and milk.Growth hormone transgenes have been inserted into genomes of pig,sheep, and cow; (2) For improved resistance to diseases - A number ofgenes contributing to the immune system (such as heavy and light chainsof an antibody that binds to a specific antigen) can be introduced to conferin vivo immunization to transgenic animals; (3) To raise transgenic animalsfor the production of pharmaceutical proteins - The concept of using farmanimals as bioreactors has raised the prospect of a revolutionary role oflivestock species. The list of proteins includes human lactoferrin, humancollagen, ttj-antitrypsin, blood coagulation factor, anticlotting agents, andothers. The prospect of producing pharmacologically active proteins in themilk of transgenic livestock is appealing for several reasons. (1) Trans-genic animals may ultimately be a low-cost method of producing recombi-nant proteins than mammalian cell culture. Lines of transgenic livestock,although are costly to establish, can be multiplied and expanded rapidlyand easily. In contrast, the maintenance of large-scale mammalian cellculture requires continuous high expense. (2) Unlike microbial systemsthat are not capable of posttranslational processing, transgenic animalsproduce bioactive complex proteins with an efficient system of post-modification. (3) Recovery and purification of active proteins from milk isrelatively simple. The volume of milk production is large, and the yield of
198 The ABCs of Gene Cloningprotein may be potentially high, rendering the process economically feasi-ble.21.1 General Procedure For Production of TransgenicAnimals In a general scheme, the gene of a desired protein is constructed ina suitable vector carrying the regulatory sequence of a milk protein whichto direct the expression in mammary tissues. Promoters that have beenused often include those of the genes of P-lactoglobulin and P-casein(major proteins found in milk). The recombinant DNA is then introducedinto the pronuclei of fertilized eggs at an early stage by microinjection.The injected DNA is usually integrated as multiple tandem copies at ran-dom locations. The transformed egg cell is then implanted into the uterusof a surrogate animal to give birth to transgenic offspring. The transgenicanimal can be raised for milking the expressed protein for processing andpurification. Stable transmission of the transgene to succeeding genera-tions is a critical factor in establishing transgenic lines of the livestock.Although it is not as frequent, transgenes can also be introduced using nu-clear transfer techniques (see Sections 22.2).21.2. Transgenic Sheep for a^-Antitrypsin The raise of transgenic sheep for the production of ttj-antitrypsinhas been described (Wright et al. 1991. Bio/Technology 9, 830-834). Hu-man tti-antitrypsin (HttjAT) is a glycoprotein with a molecular weight of54 kD, consisting of 394 amino acids, with 12% carbohydrates. The pro-tein is synthesized in the liver and secreted in the plasma with a serumconcentration of ~2 mg per ml. Human ttjAT is a potent inhibitor of awide range of serine proteases, a class of enzymes, if leave unchecked, cancause excessive tissue damage. Individuals deficient in the protein risk thedevelopment of emphysema. In the study, a hybrid gene was constructed by fusing the HttjATgene to the 5 untranslated sequence of the ovine P-lactoglobulin (PLG)gene. The HttjAT gene consisted of five exons (I, II, III, IV, and V) andfour introns. In the gene construct, the first HttjAT intron (between exonsI and II) sequence was deleted. This HttjAT minigene therefore consistedof exons I and II fused, and exons III, IV and V interrupted by introns, the
Transpharmers - Bioreactors for Pharmaceutical Products 199HttiAT initiation codon (ATG), stop codon (TAA), and polyA terminationsignal. The 5 untranslated PLG sequence included the PLG promoter, theTATA box, and the PLG exon I sequence (Fig. 20.1). (xAT exon I ^Q exon I | II III IV Promoter ^ Stop codon TATA ATG poWA) -4.0 kb -6.5 kbFig. 21.1. The hybrid gene construct of human tti-antitrypsin fused with the 5untranslated sequence of the ovine P-lactoglobulin gene. The hybrid gene construct was microinjected into sheep eggs col-lected from donor ewes following artificial ovulation and insemination.Southern blot analysis of the genomic DNA samples identified 5 trans-genic animals from 113 lambs. The transgene was shown integrated inmultiple (2-10) copies. Three of the transgenic sheep produced offspring,and these three lactating sheep were used for daily milk collection. Themilk samples were analyzed by radial immunodiffusion assay for the pres-ence of HttjAT. The milk samples were also used to purify the protein forsodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE)analysis. All three transgenic sheep produced the human protein exceed-ing 1 g per liter. The protein appeared to be glycosylated and fully active.Review1. List the advantages and disadvantages of using Hvestock animals for the pro- duction of pharmaceutical proteins.2. Why are promoters of the P-lactoglobulin and P-casein genes used for animal transgenes?3. In the example described, the transgene was integrated in multiple copies in the genome.4. Can a transgene be integrated by targeting a specific location in the chromo- some? Explain your approach.