1 Production of proteins in the of transgenic livestock: problems, solutions, and successes1 2 Alan Colman ABSTRACT The of livestock can be modified dramatically by introducing foreign DNA into the germline. Exclusive expression of this DNA is ensured by the presence of regulatory sequences from mammary gland-specific genes. n sheep > 50% of the protein in can be encoded by a transgene and it appears that the foreign protein is additional to the normal complement of proteins. However, many technical hurdles (DNA configuration, low efficiency of transgenesis, and transgene stability) still prevent the routine use of this technology. n addition, such products have not yet received regulatory approval. These difficulties are not insurmountable. Transgenic methods can also be used to study the molecular basis of biogenesis. The effect on production in mice with all endogenous a-lactalbumin genes removed and in mice in which the murine genes were then replaced with human homologues is described. a-lactalbumin, and consequently lactose, is essential for normal formation, and the human gene is expressed more efficiently than the munne gene. Am J C/in Nutr l996;63:639s-4ss. KEY WORDS Transgenic livestock, a-lactalbumin, gene knockout NTRODUCTON Genetic engineering has made it feasible to clone the proteinencoding genes from any organism. These recombinant genes can be expressed in a variety of systems ranging from simple microbial systems such as bacteria, yeast, and cultured mammalian cells to complex eukaryotic systems such as transgenic plants and animals. When the primary objective is to produce and purify a specific protein or group of proteins, the choice of system is dictated by many factors, including cost of production, amount of protein required, intended use, length of time required for development, and nature of the target proteins. As examine below, many eukaryotic secretory proteins that are complex or are required in large amounts are best made in the of transgenic animals. To date, most transgenic proteins that have been expressed in normally originate in tissues other than mammary gland. However, the most natural use of this technology must include modifying the protein content of by manipulating the -protein genes themselves. explore ideas for such a program that involve overexpression of a-lactalbumin. Finally, it is axiomatic that greater knowledge of a biological system facilitates designing experiments to manipulate the system for useful purposes. a-lactalbumin has two functions: in several species, including humans, it is an important nutritional component of and in most species it is responsible for the synthesis of lactose, which has long been implicated as the principal osmotic factor in formation (1). n the last section of this article discuss the effect on formation in mice of removing the murine a-lactalbumin gene and replacing it with its human homologue. TRANSGENC TECHNOLOGY: FUNDAMENTAL SSUES Choice of protein Most of the proteins that are targets of the biotechnology and food industries are secretory in nature and many of them assume a highly convoluted but reproducible conformation as they pass through the secretory pathway. n eukaryotic cells, many of these proteins undergo complex posttranslational modifications, leading to alterations that may be essential for functionality. t has long been known that prokaryotic systems like bacteria cannot perform most of these modifications, and that even the folding of a protein (eg, of human serum albumin) can be subtly different when the protein is made in bacteria. Even though yeast and higher plants can make many of the modifications, they are restricted in the scope of the modifications conferred; this restriction can prove unacceptable, particularly when the protein is intended for therapeutic use. A choice must be made between mammalian cell culture, with its proven track record of producing safe and effective therapeutic products, and transgenic animal technology leading to production, which is clearly superior in production yields and costs, but has yet to clear regulatory hurdles and may have some disadvantages in the length of time required for development and in consumer acceptance. Choice of transgemc species The generation times and relevant developmental events for the major domestic livestock as well as for mice and rabbits are shown in Table 1. A significant feature is the time to the birth of the G2 and G3 animals, because these times correspond to the start of lactation in what could be the production animals. The start of lactation of the G0 females is also a salient feature of these generation times because traditionally this is the earliest guide to the potential productivity of the animals and their From PPL Therapeutics Ltd, Roslin, Edinburgh, United Kingdom. 2 Address reprint requests to A Colman, PPL Therapeutics Ltd. Edinburgh EH25 9PP, United Kingdom. Am J Clin Nutr 1996:63: Printed in USA American Society for Clinical Nutrition 6395
2 6405 COLMAN TABLE 1 Livestock generation times Species G0 birth G0 adult G, birth (first ) G, adult G, birth G3 birth (G2 herd) mo Sheep or goat S  Cow  Pig Rabbit Mouse Figures in brackets refer to the time of first by using premature induction of lactation. future progeny. However, we recently obtained such predictive information much earlier in sheep by using hormonally induced, premature lactation. Nonetheless, it is clear from Table 1 that a considerable latent period exists between the beginning of a transgenic program in livestock and first, a feature made less desirable by our present inability to custom-design expression levels. Because of the unavoidable delays associated with livestock production, it is routine for transgenic programs to begin with a pilot study in mice. The pilot study can furnish valuable information on the adequacy of the design of the DNA construct and the ability of the mammary gland to produce proteins of the desired quality. The murine model, however, is not totally reliable because both protein yields and protein quality are greater from larger animals such as sheep (2) and pigs (3). The length of time to production is obviously a major factor in the choice of species. However, other considerations apply, such as the disease status of the animals, litter size, and volume of. The relative merits of each species according to a variety of criteria are shown in Table 2. Choice of DNA construct The most common method of making transgenic mammals is to inject DNA that encodes the desired protein into the pronucleus of a fertilized embryo. The embryo is transferred to a foster mother, where it continues its development to birth. Expression of the DNA in the target tissue is effected by fusing the region of DNA that encodes the protein to a regulatory DNA sequence that is known to be active in that tissue. The first transgenic mammals were made with intronless viral or mammalian DNAs (4, 5). These DNA constructs contained inappropriate or truncated regulatory sequences that did not behave in a tissue-specific way. Milk-specific expression is now effected by fusing the target gene downstream of the regulatory sequence obtained from any of a variety of protein genes, including genes encoding murine whey acidic TABLE 2 Relative merits of species Species Volume of Time to production Ease of generating founders Health Sheep Goat Cow Pig Rabbit protein (WAP), ovine /3-lactoglobulin, bovine a-lactalbumin, bovine asl-casein, and caprine (3-casein (6). Although WAPdriven genes have proved problematic in pigs (7) because of premature termination of lactation, genes driven by other protein regulatory sequences are approximately equivalent. However, the nature of the target gene configuration does seem important: a variety of experiments in mice have shown that genomic DNA is almost always superior to complementary DNA (cdna) (Table 3). This trend was clearly shown in sheep for the production of al-antitrypsin (AAT) (2, 12, 15; Table 4). Even with the availability of genomic DNA, problems still arise because of the random nature of the chromosomal integration of the foreign DNA. This randomness leads to unpredictable, and often disappointing, levels of expression. The discovery of autonomously acting and tissue-specific locus control regions (LCRs) that confer copy number-dependent expression on the target gene has led to one remedy (22). Unfortunately, no mammary gland-specific LCRs have yet been isolated. Another remedy might be to use very large pieces of DNA (>100 kb) in the form of yeast artificial chromosomes (YACs) because, in principle, these might contam the requisite LCRs even if they have not been identified formally. Transgenic animals have been made with YACs and their expression phenotypes resemble those expected for genes that are accompanied by LCRs (23). Choice of founder Founder transgenic animals usually have one or more copies of the transgene inserted into a locus on a single chromosome. These animals are therefore heterozygous for the transgene (strictly speaking, hemizygous, because there is no corresponding allele on the sister chromosome). Because the insertion process is random, every founder has a different genotype. As a result, expression levels in different founder animals that contain the same gene construct can vary widely (12). n addition, not all founders pass on any or all of their complement of transgenes intact (3, 12). The reasons for this are not completely understood, but the frequent integration of several copies of a gene at a single locus might stimulate recombination events at that locus. Furthermore, many founder animals are mosaic (not all cells or tissues are transgenic); a germ tissue that is nontransgenic could account for the lack of transmission. Because stability of inheritance and protein expression are imperative to any long-term production strategy, several founders have to be maintained and evaluated simultaneously. This situation could probably be resolved with the availability of livestock embryonic stem cells, as discussed in another article in this supplement (24). Purification and validation The criticism that followed early attempts at attracting commercial interest in the use of transgenic was that specific proteins in are difficult to purify to pharmaceutical standards at reasonable cost. We showed that these early concerns were unfounded because it proved easy to purify products such as human AAT to well greater than 99% purity. The secret is in removing the lipid, most of which is removed by low-speed centrifugation and the remainder by differential precipitation and chromatography. Purification challenges in separating
3 TRANSGENC PROTENS PRODUCED N MLK MS TABLE 3 Effect of DNA configuration on expression in transgenic mice Protein and DNA Expression in Relative transcription Reference gl RNA molecules/cell hsa cdna 0-8 gdna hpc cdna gdna haat cdna gdna haf cdna gdna rgh cdna gdna mmt-l cdna gdna hf3g cdna gdna hsa, human serum albumin; hpc, human protein C; haat, human al-antitrypsin; haf, human angiogenesis factor; rgh, rat growth hormone: mmt-l, mouse metallothionein ; hg, human j3 globin; cdna, complementary DNA; gdna, genomic DNA. highly homologous proteins remain, but these are common to all production systems. A second and more pernicious concern is related to animal health. n particular, two issues are specific to the production of proteins in animals. First, there is the issue of prions, which are responsible for neuropathic conditions like scrapie and bovine spongiform encephalopathy (BSE) in infected animals and are similar to the agent that causes Creutzfeldt-Jakob disease in humans. For sheep -produced proteins for parenteral administration, we adopted the approach of validating the punfication process for removing scrapie-producing prions and of using only animals of New Zealand origin because that country is free from the disease. n our program of producing novel bovine s for oral use, we believe that validation is not appropriate because oral ingestion of bovine or ovine has never been associated with transmission of neuropathic conditions to man; however, we do use cows from a BSE-free country, the United States. t is interesting to note that pnion removal is not validated in the human blood fractionation industry despite the possibility of contamination of pooled plasma donations with blood from persons with Creutzfeldt- Jakob disease. The second issue is that, practically speaking, it is impossible to ensure that production animals retain the same disease status from day to day. Certain viruses are endemic to particular livestock species in all parts of the world, and subclinical infections could go unnoticed. Our strategy is to identify the viruses of concern and to keep the animals virus-free if possible but, failing that, to ensure that purification would remove those viruses even if every animal in a production flock or herd were subclinically infected. Such a strategy requires information on the maximum possible viral level that could occur in the of an infected animal. We are beginning to gather this information. Not surprisingly, regulatory authorities in both the United States and Europe have been drafting guidelines for the production of transgenic livestock. The European document was finalized in December 1994, and a US Food and Drug Administration (FDA) Points to Consider was published in 1995 (25). Both of these documents concentrate on the development in transgenic animals of products for parenteral use. ssues of genetic stability and the microbiological burden of production animals figure prominently in the European document, and FDA presentations indicate similar concerns. Regarding microbiological issues, we would expect less-stringent guidelines in instances in which the products are for oral use, because there is no reason to believe that transgenic production animals would pose any special hazards. However, genetic stability will TABLE 4 Expression of human proteins in of transgenic animals Expression2 Protein, species, and DNA Modification Milk Cell culture al-antitiypsin, sheep cdna N-Glycosylation ( 2) 0.()44 (15) gdna N-Glycosylation >30 (2) (15) Factor X, sheep: cdna N-Glycosylation, y-carboxylation, proteolytic cleavage (16) <0.005 ( 17) Protein C, pig: cdna Tissue plasminogen activator, goat: cdna N-Glycosylation, y-carboxylation, proteolytic cleavage N-Glycosylation Fibrinogen, mouse: gdna N-Glycosylation, assembly > cdna, complementary DNA; gdna, genomic DNA. been 2 Reference numbers in parentheses. 3 cdna used exclusively: use of genomic sequences always gives lower yields in cell culture. (3) 3 (19) gl <0.01 ( 8) (20) 4 Recent data (21) indicate that a splicing abnormality leads to production of predominantly aberrant (nonsecretory?) protein. The errant gene has now repaired. 5 A Colman, Cottingham, Gamer, unpublished data, 1995.
4 6425 COLMAN be an issue and so will be the production of novel immunogenic proteins; the latter is under review in the United States and the United Kingdom regarding transgenic foods in general. TRANSGENC TECHNOLOGY N ACTON Overview All the published expression levels of protein from of transgenic livestock are shown in Table 4. Several different proteins of various complexity have now been expressed successfully in concentrations > gl in large animals. This contrasts with the generally much lower concentrations in mammalian cell culture. More complex proteins have now been elaborated in murine mammary glands. For example, fibrinogen is composed of six polypeptides: two a, two /3, and two y chains. These are normally assembled in liver cells into a hexamer. The secreted hexamer is cleaved in the blood by thrombin to form fibrin. Human fibrinogen secreted in the of transgenic mice in concentrations >2 g/l (Table 4) is thus far indistinguishable from protein derived from human plasma. Again, mammahan cell culture has proved poor by comparison. Production of human AAT in sheep We reported previously the generation of five founder sheep containing the human AAT gene (2). a-antitrypsin is a singlechain glycoprotein consisting of 384 amino acids that is secreted mainly from liver. ts main role is thought to be the inhibition of neutrophil-denived elastase. A common congenital deficiency of active AAT is responsible for the development of emphysema; however, because uncontrolled tissue degradation by elastase is also associated with the progression of other lung diseases like cystic fibrosis and adult respiratory distress syndrome, the provision of large quantities of AAT is likely to be medically useful. Unfortunately, before the sheep source mentioned above was available, the only source of significant amounts of therapeutically useful AAT was human plasma donations, and these cannot meet expected needs. Human AAT made in bacteria and yeast proved unsuitable and mammalian cell culture could not meet the demands for large amounts of the protein at reasonable costs. The founder females produce from 1 to 35 g AATL, and these amounts have been sustained through each of three lactations. Unfortunately, the transgenic locus in one of these animals, the highest-producing one, was not stable and her progeny all inherited different AAT copy numbers. However, the genetic locus in one of the founder males has been shown to be stable over four generations, and to date female progeny from three of these generations have produced from 13 to 18 g AAT/L consistently (12). We are now developing a production flock from a set of G, half-sisters that were all produced with the semen of the same, male of the line. t is hoped that the AAT from these animals will enter clinical trials in The data gathered in this AAT program provide the most compelling demonstration of this technology. They also address a fundamental aspect of composition: we have now analyzed the of several different animals that produce from 35 to 47 g AATL. This production does not seem to be at the expense of other proteins, which continue to be produced in normal amounts (40-50 gl), a situation that differs from that seen in overproducing transgenic mice (26). We may expect a similar situation in cows, although in the elite production animals (animals of the highest pedigree related to -producing ability) an upper limit of protein concentration may be imposed by saturation of upstream processes such as food digestion and metabolic interconversion. Nevertheless, it seems that this technology has the potential to radically remodel the protein content of. Modification of -protein content in cows Clark (24) commented on the possible benefits that could accrue if could be custom-modified in several ways. So far there is only one example of the remodeling of bovine : the generation of animals that contain the gene for human lactoferrmn (27). discuss some future nutritional targets of interest in more detail. a-lactalbumin is a small (123 amino acids), single-chain protein that is present in whey. ts prominence varies by species: in humans it is the major whey protein and in cows it is a minor whey component. Because of its well-balanced amino acid composition, a case could be made for increasing the amount of a-lacta.lbumin in infant formulas at the expense of /3-lactoglobulin (not present in human ), which has a less-suitable amino acid composition for human infants and the presence of which in bovine-derived products may contribute to allergy problems in the young. Site-directed mutagenesis is another valuable genetic engineering technique that now makes it possible to custom-design alterations of amino acids of any protein for which a gene and a suitable expression system are available. This technique can be applied to the design of special foods with therapeutic properties, one exampie being the production of novel proteins for patients with phenylketonuria (PKU). PKU is a congenital disease that results from the absence of the enzyme that metabolizes phenylalanine. Patients with PKU require a diet low in phenylalanine. This is particularly so for infants and pregnant women. Formulas low in phenylalanine are available but they are unappetizing and compliance is a problem, especially after infancy. A remedy might be available that exploits our ability to modify by transgenic means and the knowledge that a-lactalbumin contains only four phenylalanine residues. The position of each of these residues can be determined in the three-dimensional protein structure; therefore, site-directed mutagenesis with the goal of substituting other amino acids for the phenylalanine residues without disrupting the protein structure can be envisioned. The modified protein could be expressed in and then purified to provide the base for an improved diet for patients with PKU. Obviously, such purification would be helped if the endogenous a-lactalbumin content could be abrogated by either gene deletion or antisense technology. The generation of transgenic cows is labor-intensive and time-consuming. However, in many ways it is simpler than the current methods for other transgenic livestock. The most convenient method involves the in vitro maturation and fertilization of bovine oocytes that are removed from the ovaries of slaughtered animals. The fertilized embryos are then centrifuged and one of the exposed pronuclei is injected with the chosen DNA. The embryos are cultured for 7 d in vitro and the surviving embryos ( 7%) are transferred to hormonally synchronized recipients. The pregnancy rate we obtain is 20%.
5 TRANSGENC PROTENS PRODUCED N MLK 6435 Published data indicate a transgenic integration rate of 5% (27), so roughly 5 of every 100 live births can be expected to be transgenic. At best, one transgenic animal for about every 1500 embryos injected can be expected. Fortunately, we are now finding it possible to inject >800 embryos per week. The limiting factor-and also the most expensive-is the number of appropriate recipients required. However, because no invasive procedure is ever used, the recipients can be recycled. This contrasts with the present manufacture of other transgenic livestock animals, in which surgery is required for both the embryo donors and the recipients, thereby limiting the recycling options. Marginal improvements in the process can already be brought about (at great cost) by, for example, the repeated use of live cow embryo donors (such embryos develop better). However, significant improvements in the efficiency of the transgenic process will be made only when injected embryos can be screened for transgene integration before their transfer to recipients, when the integration rate can be improved, or, best of all, when transformable embryonic stem cells become available. The chronology of a -modification program using the present technology is shown in Figure 1. Significant features of the program are that a high-expressing G0 male or female founder makes collection from a modest production herd possible within 5 y. However, for this to occur in a costeffective way, nonroutine procedures such as premature induction of lactation of bulls and transvaginal oocyte recovery from cows are required. Large herds of animals can be expected after 6.5 y. Herd development would be expedited by the generation of homozygous animals (ie, animals in which the transgenes are present on both sister chromosomes) because all female offspring of these animals would be transgenic. However, it would be 9.5 y before these herds would be available for ing. a-lactalbumin and formation t has been argued above that in certain situations the transgenic mammary gland is superior to the cultured mammalian cell as a protein-production system. However, the expression of added genes in the mammary gland may have unwelcome effects on both mammary gland function and the physiology of the organism as a whole. a-lactalbumin may exert a strong influence on carbohydrate and fluid content through its role as a component of the lactose synthase complex. t associates with galactosyltransferase, a Golgi membrane protein, to change the substrate specificity of that enzyme and promote lactose synthesis. n many species, lactose is thought to be the major osmotic influence in determining volume. There is a correlation between lactose, fluid, and a-lactalbumin concentrations in the s of different species, but no causative relation has yet been established (28). f formation is indeed sensitive to a-lactalbumin synthesis, this could affect some of the programs suggested above. For example, overexpression of native a-lactalbumin could lead to extra lactose formation and a more dilute, and an overexpressed, mutated a-lactalbumin might be a competitive inhibitor of lactose synthase and reduce lactose synthesis, resulting in a highly concentrated. Clearly, these issues could be resolved empirically by performing overexpression experiments and analyzing the results. Female birth G0 Mature breed G1 births Test herd G0 Milk Milk (induced) G1 Male homozygous Homozygous males Birth of Backcrose males/females herd Birth G2s of production G2s of producherd tion Milk Male G0 Mature female birth breed births - (induced)? G1 Gl Male Milk ( induced) G1 herd (production)? Homozygous homozygous BackcroBa males/females males Birth herd of herd of production Birth G2 of production G2s Months FGURE 1. Time required to produce a ing herd of transgenic cows. starting from the injection of DNA into a one-cell embryo.
6 6445 COLMAN However, a more fundamental understanding of the role of a-lactalbumin in formation would undoubtedly be beneficial to the experimental design. We conducted experiments in which we generated mice with no murine a-lactalbumin genes and mice in which the murine a-lactalbumin genes were replaced by human a-lactalbumin genes (29). A description of the phenotypes of mice that contained different combinations of the a-lactalbumin genes from the two species follows; for the original data refer to the study by Stacey et al (30). The a-lactalbumin concentrations in the s of the different mouse lines were estimated by direct visualization on polyacrylamide gels as well as by quantitative adsorption chromatography with phenyl-sepharose columns. A normal (wildtype) mouse is designated alacm/alacm; this designation refers to the presence (alacm) or absence (alac) of the murine (m) a-lactalbumin gene on each sister chromosome. Wild-type mice (alacm/alacm) have only a small amount of a-lactalbumin in their (0.9 gl) and this concentration is halved in heterozygous mice with only one copy of the gene (alacm/ alac). Nevertheless, the composition of the two s is similar and even the small reduction in lactose in the heterozygotes is not significant. These data indicate that, although synthesis of a-lactalbumin is dependent on gene dose, a-lactalbumin concentrations in the cell are not limiting for lactose synthesis. t is of little surprise that mice with no copies of the a-lactalbumin genes (alac/alac) make no a-lactalbumin and their is radically different from that of the mice discussed above. t is viscous and extremely protein- and lipid-rich and contains no lactose. Milk volumes in mice with no copies of the gene are dramatically reduced and these animals are unable to rear litters successfully, but all other developmental functions appear normal. We conclude that the presence of a-lactalbumin is essential to lactose formation and that this in turn drives volume. The content of a-lactalbumin relative to that of other proteins is considerably greater in human (16-25%) than in mouse ( 0.l%). This difference is maintained when the human gene is substituted for the mouse gene. The human replacement gene is designated alach. Humanized (alach/alach) mice secrete 1.4 mg a-lactalbumin/l. We showed by using heterozygous (alacm/alach) mice that this increased secretion is due to a higher rate of transcniption that occurs on the human gene. Milk from totally humanized mice is similar to that from wild-type mice except that the volumes are greater in humanized mice. We conclude that human a-lactalbumin can functionally substitute for the mouse protein in the mouse mammary gland. We speculate that the increased volume is due to increased lactose formation that results from either the surplus of a-lactalbumin protein or improved kinetic properties of the hybrid lactose synthase. CONCLUSONS Transgenic technology can be used to both exploit the impressive productivity of the mammary gland and provide information about the molecular mechanisms that underlie that productivity. Large amounts of a desired protein can be obtamed from the of transgenic animals. The remaining major hurdles involve increasing the efficiency of generating transgenic animals, successfully addressing regulatory issues, and, particularly in the area of modified foods, maintaining the low cost of large-scale purification and overcoming public concerns. A REFERENCES 1. Kuhn Ni. The biosynthesis of lactose. n: Mepham TB, ed. Biochemistry of lactation. Amsterdam: Elsevier, 1983: Wright G, Carver A, Cottom D, et al. High-level expression of active human aipha-l-antitrypsin in the of transgenic sheep. Biotechnology (N Y) 199l;9: Velander WH, iohnson JL, Page RL, et al. High-level expression of a heterologous protein in the of transgenic swine using the cdna encoding human protein C. Proc Natl Acad Sci U S A l992;89: Gordon JW, Scangos GA, Plotkin Di, Barbosa JA, Ruddle FH. Genetic transformation of mouse embryos by microinjection of purified DNA. Proc Natl Acad Sci U S A l980;77: Wagner TE, Hoppe PC, iollick JD, Scholl DR. Hodinka RL, Gault JB. Microinjection of a rabbit 3-globin gene into zygotes and its subsequent expression in adult mice and their offspring. Proc Nat Acad Sci U S A 1981;78: Houdebine L-M. Production of pharmaceutical proteins from transgenic animals. Biotechnol 1994;34: Wall Ri, Pursel VG, Shamay A, McKnight RA, Pittius CW, Hennighausen L. High-level synthesis of a heterologous protein in the mammary glands of transgenic swine. Proc Natl Acad Sci U S A 1991 ;88: Shani M, Barash, Nathan M, et al. Expression of human serum albumin in the of transgenic mice. Transgenic Res 1992:1: Velander WH, Page RL, Morcdl T, et al. Production of biologically active human protein C in the of transgenic mice. Ann N Y Acad Sci 1992;665:39l Drohan WN, Zhang DW, Paleyanda RK, et al. nefficient processing of human protein C in the mouse mammary gland. Transgenic Res 1994;3: Whitelaw CBA, Archibald AL, Harris 5, McClenaghan M, Simons JP, Clark AJ. Targeting expression to the mammary gland: intronic Sequences can enhance the efficiency of gene expression in transgenic mice. Transgenic Res 1991 ; : Carver AS, Dalrymple MA, Wright G, et al. Transgenic livestock as bioreactors: stable expression of human alpha- -antitrypsin by a flock of sheep. Biotechnology (N Y) 1993; 1 1: Palmiter RD. Sandgren EP, Avarbock MR. Allen DD, Brinster RL. Heterologous introns can enhance expression of transgenes in mice. Proc Nat Acad Sci U S A 1991;88: Brinster RL, Allen JM, Behringer RR, Gelinas RE, Palmiter RD. ntrons increase transcriptional efficiency in transgenic mice. Proc Nati Acad Sci U S A 1988:85: Paterson T, nnes, Moore S. Approaches to maximizing stable expression of a1-antitrypsin in transformed CHO cells. Appl Microbiol Biotechnol 1994;40:69l Clark Al, Bessos H, Bishop JO, et al. Expression of human antihemophilic factor X in the of transgenic sheep. Biotechnology (N Y) l989;7: Anson DS, Austen DEG, Brownlee GG. Expression of active human clotting factor X from recombinant DNA clones in mammalian cells. Nature l985;3 15: Foster DC, Sprecher CA, Holly RD. Gambee JE, Walker KM. Kumar AA. Endoproteolytic processing of the dibasic cleavage site in the human protein C precursor in transfected mammalian cells: effect of sequence alterations on efficiency of cleavage. Biochemistry 1 990;29:
7 TRANSGENC PROTENS PRODUCED N MLK 645S 19. Ebert KM, Selgrath ip, DiTullio P, et al. Transgenic production of a variant of human tissue-type plasminogen activator in goat : generation of transgenic goats and analysis of expression. Biotechnology (N Y) l991;9: Datar RV, Cartwright T, Rosen C-G. Process economics of animal cell and bacterial fermentations: a case study analysis of tissue plasminogen activator. Biotechnology (N Y) 1993;ll: Yull F, Harold G, Wallace R, et al. Fixing human factor X (fix): correction of a cryptic RNA splice enables the production of biologically active fx in the mammary gland of transgenic mice. Proc Natl Acad Sci U S A 1995: Grosveld F, van Assendelft GB, Greaves DR. Kollias G. Positionindependent, high-level expression of the human /3-globin gene in transgenic mice. Cell l987;5l: Schedl A, Montoliu L, Kelsey G, Schtitz G. A yeast artificial chromosome covering the tyrosinase gene confers copy number-dependent expression in transgenic mice. Nature l993;362:258-6l. 24. Clark Al. Genetic modification of proteins. Am Clin Nutr 1996;63:633S-8S. 25. Food and Drug Administration. Points to consider in the manufacture and testing of therapeutic products for human use derived from transgenic animals. FDA Center for Biologics Evaluation and Research, Wilde Ci, Clark Al, Kerr MA, Knight CH, McClenaghan M, Simons JP. Mammary development and secretion in transgenic mice expressing the sheep (3-lactoglobulin gene. Biochem 1992;284:7l Krimpenfort P, Rademakers A, Eyestone W, et al. Generation of transgenic dairy cattle using in vitro embryo production. Biotechnology (N Y) 199 l;9: Fitzgerald DK, Brodbeck U, Kiyosawa, Mawal R, Colvin B, Ebner KE. a-lactalbumin and the lactose synthetase reaction. Biol Chem l970;245: Stacey A, Schnieke A, McWhir, Cooper, Colman A, Mellon DW. Use of a double-replacement gene targeting to replace the murine a-lactalbumin gene with its human counterpart in embryonic stem cells and mice. Mol Cell Biol 1994;l4:1009-l Stacey A, Schnieke A, Kerr M, et al. Lactation is disrupted by a-lactalbumin deficiency and can be restored by human a-lactalbumin gene replacement in mice. Proc Nail Acad Sci U S A 1995;92:
Transgenic Animals: Their Benefits To Human Welfare Endang Tri Margawati An Action Bioscience.org original article January 2003 Transgenic mice, rats, rabbits, pigs, sheep, and cows have already been created.
Transgenic technology in the production of therapeutic proteins Transgenic technology represents a new generation of biopharmaceutical production system to meet the medical needs of the new millennium.
USE OF TRANSGENIC ANIMALS IN THE MANUFACTURE OF BIOLOGICAL MEDICINAL PRODUCTS FOR HUMAN USE Guideline Title Use Of Transgenic Animals In The Manufacture Of Biological Medicinal Products For Human Use Legislative
Animal Pharming: The Industrialization of Transgenic Animals December 1999 Animal pharming, the process of using transgenic animals to produce human drugs, is staking its claim in a lucrative world market.
1. A recombinant DNA molecules is one that is a. produced through the process of crossing over that occurs in meiosis b. constructed from DNA from different sources c. constructed from novel combinations
DNA Fingerprinting Unless they are identical twins, individuals have unique DNA DNA fingerprinting The name used for the unambiguous identifying technique that takes advantage of differences in DNA sequence
Name: Period: Date: I. OVERVIEW OF GENETIC ENGINEERING: Biotechnology refers to technology used to DNA. The procedures are often referred to as. is the genetic material of all living organisms. o All organisms
What is Biotechnology? Student answers: Biotechnology Definition the science of altering genetic and reproductive processes in plants and animals What is Genetic Engineering? Student answers Genetic Engineering
Chapter 9 Biotechnology and Recombinant DNA Biotechnology and Recombinant DNA Q&A Interferons are species specific, so that interferons to be used in humans must be produced in human cells. Can you think
Name: 2.5 Genetics Objectives 2.5.9 Genetic Engineering 1. Define Genetic Engineering 2. Understand that GE alters DNA 3. Understand the function of restriction enzymes 4. Be able to explain the following
How DNA Molecules Are Analyzed Chapter 10 Manipulating Genes Until the development of recombinant DNA techniques, crucial clues for understanding how cell works remained lock in the genome. Important advances
Genetics Module B, Anchor 3 Key Concepts: - An individual s characteristics are determines by factors that are passed from one parental generation to the next. - During gamete formation, the alleles for
Growth of Animal Cells in Culture The ability to study cells depends largely on how readily they can be grown and manipulated in the laboratory. Although the process is technically far more difficult than
Biotechnology and reporter genes Here, a lentivirus is used to carry foreign DNA into chickens. A reporter gene (GFP)indicates that foreign DNA has been successfully transferred. Recombinant DNA continued
RECOMBINANT DNA TECHNOLOGY By; Dr. Adeel Chaudhary 2 nd yr Molecular Genetics Medical Technology College of Applied Medical Sciences Recombinant DNA is a form of artificial DNA that is made through the
European Medicines Agency July 1996 CPMP/ICH/139/95 ICH Topic Q 5 B Quality of Biotechnological Products: Analysis of the Expression Construct in Cell Lines Used for Production of r-dna Derived Protein
Common Course Topics Biology 1406: Cell and Molecular Biology 1. Introduction to biology --the scientific study of organisms --properties of life --assumptions, methods and limitations of science --underlying
GENETICS OF BACTERIA AND VIRUSES 1 Genes of bacteria are found in bacterial chromosomes Usually a single type of chromosome May have more than one copy of that chromosome Number of copies depends on the
Controls Over Genes Chapter 15 Impacts, Issues: Between You and Eternity Mutations in some genes predispose individuals to develop certain kinds of cancer; mutations in BRAC genes cause breast cancer normal
Control of Gene Expression (Learning Objectives) Explain the role of gene expression is differentiation of function of cells which leads to the emergence of different tissues, organs, and organ systems
Recombinant DNA and Biotechnology Chapter 18 Lecture Objectives What Is Recombinant DNA? How Are New Genes Inserted into Cells? What Sources of DNA Are Used in Cloning? What Other Tools Are Used to Study
Gene mutation and molecular medicine Chapter 15 Lecture Objectives What Are Mutations? How Are DNA Molecules and Mutations Analyzed? How Do Defective Proteins Lead to Diseases? What DNA Changes Lead to
Genetics Test Biology I Multiple Choice Identify the choice that best completes the statement or answers the question. 1. Avery s experiments showed that bacteria are transformed by a. RNA. c. proteins.
C1. A gene pool is all of the genes present in a particular population. Each type of gene within a gene pool may exist in one or more alleles. The prevalence of an allele within the gene pool is described
Course Curriculum for Master Degree in Food Science and Technology/ Department of Nutrition and Food Technology The Master Degree in Food Science and Technology / Department of Nutrition and Food Technology,
1. The transfer of genes from parents to their offspring is known as 5. The diagram below represents a reproductive process that takes place in humans. (1) differentiation (2) heredity (3) immunity (4)
Name Period _ Regents Biology Date _ REVIEW 5: GENETICS 1. Chromosomes: a. Humans have 46 chromosomes, or _23 _ homologous pairs. Homologous: _Chromosomes of the same position and size b. Chromosome pairs
How to construct transgenic mice Sandra Beer-Hammer Autumn School 2012 Bad Schandau Pharmakologie und Experimentelle Therapie (APET) Overview History Generation of embryonic stem (ES) cell lines Generation
Genetics (20%) Sample Test Prep Questions Grade 7 (2a Genetics) Students know the differences between the life cycles and reproduction methods of sexual and asexual organisms. (pg. 106 Science Framework)
BioBoot Camp Genetics BIO.B.1.2.1 Describe how the process of DNA replication results in the transmission and/or conservation of genetic information DNA Replication is the process of DNA being copied before
MUTATION, DNA REPAIR AND CANCER 1 Mutation A heritable change in the genetic material Essential to the continuity of life Source of variation for natural selection New mutations are more likely to be harmful
1 Lecture 5 Mutation and Genetic Variation I. Review of DNA structure and function you should already know this. A. The Central Dogma DNA mrna Protein where the mistakes are made. 1. Some definitions based
Biotechnology and Recombinant DNA (Chapter 9) Lecture Materials for Amy Warenda Czura, Ph.D. Suffolk County Community College Primary Source for figures and content: Eastern Campus Tortora, G.J. Microbiology
Biotechnology bio and technology The use of living organisms to solve problems or make useful products. Biotechnology has been practiced for the last 10,000 years. Selective breeding Use of microbes (bacteria
Chapter 18 Regulation of Gene Expression 18.1. Gene Regulation Is Necessary By switching genes off when they are not needed, cells can prevent resources from being wasted. There should be natural selection
Common Course Topics Biology 1414: Introduction to Biotechnology I Assumptions Students may be enrolled in this course for several reasons; they are enrolled in the Biotechnology Program, they need a science
Quality and Safety Evaluation of Gene Therapy Products in Japan Review Mechanism for Gene Therapy in Japan The review mechanism for gene therapy in Japan was partially amended to simplify the necessary
Stem Cell Quick Guide: Stem Cell Basics What is a Stem Cell? Stem cells are the starting point from which the rest of the body grows. The adult human body is made up of hundreds of millions of different
Gene Therapy and Genetic Counseling Chapter 20 What is Gene Therapy? Treating a disease by replacing, manipulating or supplementing a gene The act of changing an individual s DNA sequence to fix a non-functional
Bio 100 DNA Technology 1 Chapter 12 - DNA Technology Among bacteria, there are 3 mechanisms for transferring genes from one cell to another cell: transformation, transduction, and conjugation 1. Transformation
CHAPTER 6: RECOMBINANT DNA TECHNOLOGY YEAR III PHARM.D DR. V. CHITRA INTRODUCTION DNA : DNA is deoxyribose nucleic acid. It is made up of a base consisting of sugar, phosphate and one nitrogen base.the
Advanced Biology Notes: Human Disorder Pedigree analysis: Our understanding of Mendelian inheritance in humans is based on the analysis of family pedigrees or the results of mating that have already occurred.
Recombinant DNA technology (genetic engineering) involves combining genes from different sources into new cells that can express the genes. Recombinant DNA technology has had-and will havemany important
Manufacturing process of biologics K. Ho Afssaps, France 2011 ICH International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use 2011 ICH 1 Disclaimer:
Cell Line Data Pack Cell Line: HD-BIOP3 Suspension adapted GS null CHO K1 cells t + 44 ()1223 65558 f + 44 ()1223 655581 e firstname.lastname@example.org w www.horizondiscovery.com Horizon Discovery, 71 Cambridge
I. Genetic Engineering modification of DNA of organisms to produce new genes with new characteristics -genes are small compared to chromosomes -need methods to get gene-sized pieces of DNA -direct manipulation
PowerPoint Lecture Presentations prepared by Mindy Miller-Kittrell, North Carolina State University C H A P T E R 8 Recombinant DNA Technology The Role of Recombinant DNA Technology in Biotechnology Biotechnology
Answers to Mendelian genetics questions BI164 Spring, 2007 1. The father has normal vision and must therefore be hemizygous for the normal vision allele. The mother must be a carrier and hence the source
Human genetics: Why? Human Genetics Introduction Determine genotypic basis of variant phenotypes to facilitate: Understanding biological basis of human genetic diversity Prenatal diagnosis Predictive testing
7.03 Fall 2003 1 of 6 1. a) Genetic properties of gln2- and gln 3-: Answer Key Problem Set 5 Both are uninducible, as they give decreased glutamine synthetase (GS) activity. Both are recessive, as mating
Name: 8931-1 - Page 1 1) The diagram below represents one possible immune response that can occur in the human body. 5) One similarity between cell receptors and antibodies is that both A) are involved
Biotechnology and Recombinant DNA Recombinant DNA procedures - an overview Biotechnology: The use of microorganisms, cells, or cell components to make a product. Foods, antibiotics, vitamins, enzymes Recombinant
Expression and Purification of Recombinant Protein in bacteria and Yeast Presented By: Puspa pandey, Mohit sachdeva & Ming yu DNA Vectors Molecular carriers which carry fragments of DNA into host cell.
Genetic Disorders During Meiosis Karyotypes Genetic Technologies Learning goals Understand the errors that can occur during meiosis and identify some disorders using karyotypes Understand Mendel s 2 laws
Chapter 18 Regulation of Gene Expression PowerPoint Lecture Presentations for Biology Eighth Edition Neil Campbell and Jane Reece Lectures by Chris Romero, updated by Erin Barley with contributions from
Transformation Making Change Happen Genetic Engineering Definition: The alteration of an organism s genetic, or hereditary, material to eliminate undesirable characteristics or to produce desirable new
STUDIES ON SEED STORAGE PROTEINS OF SOME ECONOMICALLY MINOR PLANTS THESIS SUBMITTED FOR THE DEGREB OF DOCTOR OF PHILOSOPHY (SCIENCE) OF THE UNIVERSITY OF CALCUTTA 1996 NRISINHA DE, M.Sc DEPARTMENT OF BIOCHEMISTRY
Core Category Nature of Genetic Material Nature of Genetic Material Core Concepts in Genetics (in bold)/example Learning Objectives How is DNA organized? Describe the types of DNA regions that do not encode
Trasposable elements: P elements In 1938 Marcus Rhodes provided the first genetic description of an unstable mutation, an allele of a gene required for the production of pigment in maize. This instability
Proceedings, Applied Reproductive Strategies in Beef Cattle September 11 and 12, 2007, Billings, Montana NEW TECHNOLOGIES FOR REPRODUCTION IN CATTLE George E. Seidel, Jr. Animal Reproduction and Biotechnology
Introductory genetics for veterinary students Michel Georges Introduction 1 References Genetics Analysis of Genes and Genomes 7 th edition. Hartl & Jones Molecular Biology of the Cell 5 th edition. Alberts
Reproductive technologies Lecture 15 Introduction to Breeding and Genetics GENE 251/351 School of Environment and Rural Science (Genetics) Animal Breeding in a nutshell Breeding objectives Trait measurement
How to construct transgenic mice Sandra Beer-Hammer Autumn School 2011 Bad Schandau Pharmakologie und Experimentelle Therapie (APET) Overview History Generation of embryonic stem (ES) cell lines Generation
Lecture 3: Mutations Recall that the flow of information within a cell involves the transcription of DNA to mrna and the translation of mrna to protein. Recall also, that the flow of information between
KMS-Specialist & Customized Biosimilar Service 1. Polyclonal Antibody Development Service KMS offering a variety of Polyclonal Antibody Services to fit your research and production needs. we develop polyclonal
Gene Regulation -- The Lac Operon Specific proteins are present in different tissues and some appear only at certain times during development. All cells of a higher organism have the full set of genes:
1 of 7 9/7/2012 4:45 PM History of Agricultural Biotechnology: How Crop Development has Evolved By: Wieczorek Ania (Dept of Tropical Plant and Soil Sciences, University of Hi at Manoa) & Wright Mark (Dept
Human Cloning The Science and Ethics of Transplantation Rudolf Jaenisch, M.D. In addition to the moral argument against the use of somatic-cell nuclear for the creation of a child ( reproductive cloning
Genetic Improvement and Crossbreeding in Meat Goats Lessons in Animal Breeding for Goats Bred and Raised for Meat Will R. Getz Fort Valley State University Appendix J. Genetic Implications of Recent Biotechnologies
Protein Structure Polypeptide: Protein: Therefore: Example: Single chain of amino acids 1 or more polypeptide chains All polypeptides are proteins Some proteins contain >1 polypeptide Hemoglobin (O 2 binding
prosci-inc.com Custom Antibody Services High Performance Antibodies and More Broad Antibody Catalog Extensive Antibody Services CUSTOM ANTIBODY SERVICES Established in 1998, ProSci Incorporated is a leading
13.4 Gene Regulation and Expression Lesson Objectives Describe gene regulation in prokaryotes. Explain how most eukaryotic genes are regulated. Relate gene regulation to development in multicellular organisms.
G e n e C o p o eia TM Expressway to Discovery APPLICATION NOTE Introduction GeneCopoeia Genome Editing Tools for Safe Harbor Integration in Mice and Humans Ed Davis, Liuqing Qian, Ruiqing li, Junsheng
Chapter 25: Population Genetics Student Learning Objectives Upon completion of this chapter you should be able to: 1. Understand the concept of a population and polymorphism in populations. 2. Apply the
GENE CLONING AND RECOMBINANT DNA TECHNOLOGY What is recombinant DNA? DNA from 2 different sources (often from 2 different species) are combined together in vitro. Recombinant DNA forms the basis of cloning.
Gene Therapy The use of DNA as a drug Edited by Gavin Brooks BPharm, PhD, MRPharmS (PP) Pharmaceutical Press Contents Preface xiii Acknowledgements xv About the editor xvi Contributors xvii An introduction
Chapter 5: Organization and Expression of Immunoglobulin Genes I. Genetic Model Compatible with Ig Structure A. Two models for Ab structure diversity 1. Germ-line theory: maintained that the genome contributed
The world of non-coding RNA Espen Enerly ncrna in general Different groups Small RNAs Outline mirnas and sirnas Speculations Common for all ncrna Per def.: never translated Not spurious transcripts Always/often
Limited A student performing at the Limited Level demonstrates a minimal command of Ohio s Learning Standards for Biology. A student at this level has an emerging ability to describe genetic patterns of