Situación Actual

With the development of transgenic technology, improvements in commercially important livestock species have become possible by transferring genes from related or unrelated species. Genetic improve-

ment through biotechnology can be achieved in one generation, instead of the several generations re- quired for traditional animal breeding methods. Al- though several methods of gene transfer have been developed, four methods are used today in the pro- duction of most transgenic animals: nuclear transfer, microinjection, viral vector infection, and embryon- ic stem cell transfer. The nuclear transfer method entails inserting the entire genetic material from the nucleus of a donor cell into a mature unfertilized egg whose nucleus has been removed. After that, the embryo is transferred into a foster mother, where it will develop into an animal that is genetically identi- cal to the donor cell (Wolf et al. 2001). In micro- injection, a segment of foreign DNA carrying one or more genes is injected into the male pronucleus of a fertilized egg. The egg needs to be in a single-cell stage to ensure that all somatic cells in the animal contain the transgene. The embryo is then trans- ferred to the uterus of a surrogate mother (Wall 2002). In the retroviral infection technique, the gene is transferred with the help of a viral vector. Ret- roviruses are frequently used in the process of DNA transfer due to their natural ability to infect cells (Cabot et al. 2001). In the stem cell transfer tech- nique, embryonic stem cells are collected from

Figure 3.9. Biosynthetic pathway for the soybean isoflavonoids: daidzein and genistein. (Adapted from Jung et al. 2000.)

blastocysts and grown in culture. The cultured cells are then injected into the inner cell mass of an em- bryo in the blastocyst stage, which is then implanted into the foster mother, resulting in the production of a chimeric animal (Hochedlinger and Jaenisch 2003). It is important to remember that these meth- ods do not create new species; they only offer tools for producing new strains of animals that carry nov- el genetic information. Some examples of genetical- ly engineered animals include transgenic cows that produce milk with improved composition and trans- genic swine that produce meat with lower fat con- tent. The main goal of livestock genetic engineering programs is to increase production efficiency while delivering healthier animal food products.

MODIFIEDMILK INTRANSGENICDAIRY CATTLE

Bovine milk has been described as an almost perfect food because it is a rich source of vitamins, calcium, and essential amino acids (Karatzas and Turner 1997). Some of the vitamins found in milk include vitamins A, B, C, and D. Milk has greater calcium content than any other food source, and daily con- sumption of two servings of milk or other dairy products supplies all the calcium requirements of an adult person (Rinzler et al. 1999). Caseins represent about 80% of the total milk protein and have high nutritional value and functional properties (Brophy et al. 2003). The caseins have a strong affinity for cations such as calcium, magnesium, iron, and zinc. There are four types of naturally occurring caseins in milk: S1, S2, , and  (Brophy et al. 2003). They are clumped in large micelles, which deter- mine the physicochemical properties of milk. Even small variations in the ratio of the different caseins influence micelle structure, which in turn can change the milk’s functional properties. The amount of caseins in milk is an important factor for cheese manufacturing, since greater casein content results in greater cheese yield and improved nutritional quality (McMahon and Brown 1984). It has been estimated that enhancing the casein content in milk by 20% would result in an increase in cheese pro- duction, generating an additional $190 million/year for the dairy industry (Wall et al. 1997). Dairy cattle have only one copy of the genes that encode  (s1/s2), _{, and -casein proteins, and out of the four} caseins,  and  are the most important (Bawden et al. 1994). Increased milk -casein content reduces

the size of the micelle, resulting in improved heat stability. -caseins are highly phosphorylated and bind to calcium phosphate, thus influencing milk calcium levels (Dalgleish et al. 1989, Jimenez Flores and Richardson 1988).

Research on modification of milk composition to improve nutritional or functional properties has been mostly done in transgenic mice. Mice are good models for the study of protein expression in mam- mary glands, but they do not always reflect the same protein expression levels as ruminants (Colman 1996). Brophy et al. (2003), using nuclear transfer technology, produced transgenic cows carrying extra copies of the genes CSN2 and CSN3, which encode bovine - and -caseins, respectively. Genomic clones containing CSN2 and CSN3 were isolated from a bovine genomic library. Previous studies con- ducted with mice revealed that CSN3 had very low expression levels (Persuy et al. 1995). In order to enhance expression of CSN3, the researchers created a CSN2/3 fusion construct, in which the CSN3 gene was fused with the CSN2 promoter. The CSN2 genomic clone and the CSN2/3 fusion construct were co-transfected into bovine fetal fibroblast (BFF) cells, where the two genes showed coordinat- ed expression. The transgenic cells became the donor cells in the process of nuclear transfer, gener- ating nine fully healthy and functional cows. Over- expression of CSN2 and CSN2/3 in the transgenic cows resulted in an 8–20% increase in_{-casein and} a 100% increase in -casein levels (Brophy et al. 2003).

INCREASEDMUSCLEGROWTH INCATTLE

Myostatin, also known as growth and differentiation factor 8 (GDF-8), is a member of the transforming growth factor  (TGF-) family, which is responsi- ble for negative regulation of skeletal muscle mass in mice, cattle, and possibly other vertebrates. Myo- statin is expressed in embryo myoblasts and devel- oping adult skeletal muscle; it is produced as a 375- amino-acid precursor molecule that is further processed by enzymatic cleavage of the N-terminus prodomain segment. The remaining C-terminus 109-amino-acid segment is the myostatin protein (Gleizes et al. 1997). The processed protein forms dimers that are biologically active. McPherron and Lee (1997), through alignment of myostatin amino acid sequences from baboon, bovine, chicken, hu- man, murine, ovine, porcine, rat, turkey, and zebra-

fish, determined that the myostatin gene is highly conserved among vertebrates, which suggests that its function may be conserved as well. The re- searchers also demonstrated that the increase in muscle mass (double-muscling) phenotype observed in some breeds of cattle, such as Belgian Blue and Piedmontese, is due to mutations in the myostatin gene. In myostatin-null mice that had the myo- statin gene knocked out by gene targeting, individ- ual muscles weighed on average two to three times more than those in wild-type mice, and body weight was 30% higher. This difference was not due to an increase in fat amount, but to an increase in the cross-sectional area of the muscle fibers (hypertro- phy) and an increase in the number of muscle fibers (hyperplasia) (McPherron and Lee 1997).

It is believed that myostatin prodomain may bind noncovalently with the mature myostatin, resulting in inhibition of the biological activity of myostatin (Thies et al. 2001). Based on the general molecular model of TGF proteins, Yang et al. (2001) hypothe- sized that overexpression of the prodomain segment would interfere with mature myostatin, resulting in the promotion of muscle development. The myosta- tin prodomain DNA was cloned into a pMEX- NMCS2 vector, which contained a rat myosin light chain 1 (MLC1) promoter, a SV40 poly adenylation sequence, and a MLC enhancer. This construct was then inserted into the mouse genome using the pronuclear microinjection technique. In comparison with wild-type mice, overexpression of the myo- statin prodomain in the transgenic mice resulted in a 17–30% increase in body weight; a 22–44% in- crease in total carcass weight at 9 weeks of age (Fig. 3.10); and a significant decrease in epididymal fat pad weight, which is an indicator of body fat mass (Yang et al. 2001). No undesirable phenotypes or health or reproductive problems were observed in the transgenic animals. These results indicate that this same approach can be used to develop farm ani- mals with enhanced growth performance, increased muscle mass, and decreased fat content, which would equate to a healthier meat product for consumers (Yang et al. 2001).

REDUCEDFATCONTENT INTRANSGENIC SWINE

In the human diet, ingested exogenous fats serve as the raw material for the synthesis of fat, cholesterol, and many phospholipids. Since fat energy content is

two times greater than the energy obtained from car- bohydrates and proteins, most of the energy that is stored in the body is in the form of fat. Fats are a group of chemical compounds that contain fatty acids. The most common fatty acids found in animal fats are palmitic acid, stearic acid, and oleic acid. The human body is able to synthesize these fats, but there is one more class of fatty acids called the essential fatty acids (linolenic acid, linoleic acid, and arachidonic acid), which the body cannot pro- duce; they therefore must be obtained from diet (Campbell and Reece 2002). There are two main types of naturally occurring fatty acids: saturated and unsaturated. Saturated fatty acids (SFA) are mainly animal fats. They are called saturated because all the carbon chains are completely filled with hydrogen and there are no double bonds formed between the carbon atoms. Saturated fatty acids are believed to be “bad” fats since they raise both high-density lipoprotein (HDL) and low-densi- ty lipoprotein (LDL) cholesterol (Keys et al. 1965, NRC 1988). Unsaturated fats are found mainly in products derived from plant sources and are divided into two categories: monounsaturated fatty acids (MUFA), which have one double bond; and polyun- saturated fatty acids (PUFA), which have two or more double bonds in the carbon chain. It has been observed that the increased consumption of these “good” fats actually reduces LDL levels and en- hances HDL levels (Grundy 1986, NRC 1988). It is

Figure 3.10. Comparison between wild-type and trans- genic mice overexpressing myostatin prodomain. (Courtesy of Dr. J. Yang, Univ. of Hawaii.)

now well established that a high fat diet (specially of SFA) not only increases the risk of heart disease but also the risk of breast, colon, and prostate cancer. Many health agencies, including the American Die- tetic Association, the American Diabetes Associ- ation, and the American Heart Association, recom- mend that fat intake should be no more than 30% of the total daily calories.

In research conducted by the United State De- partment of Agriculture (USDA), scientists intro- duced a recombinant bovine growth hormone (rBGH) gene into pigs, with the purpose of under- standing the relationship between rBGH expression and the amount of fatty acids in the animal (Solomon et al. 1994). Bovine growth hormone (BGH), also known as bovine somatotropin, which is produced in the pituitary gland, stimulates growth in immature cattle and enhances milk production in lactating cows (Leury et al. 2003). BGH is a protein hormone, and as such, it is broken down during digestion in the gastrointestinal tract, making it biologically inactive in humans (Etherton 1991). In 1993, based on rigor- ous scientific investigations, the U.S. Food and Drug Administration (FDA) concluded that products from transgenic-BGH and supplemented-BGH animals are safe for human consumption.

Bovine growth hormone has been shown to decrease fat content of transgenic pigs expressing an rBGH gene (Pursel et al. 1989). The transgenic pigs used in this study were created by pronuclear micro- injection technique. The gene encoding rBGH was introduced into the pig genome under the control of the mouse metallothionein-I (MT) promoter. After rBGH transgenic lines of pigs were established, suc- cessive generations were produced by artificial in- semination of nontransgenic females with sperm collected from rBGH transgenic males. To deter- mine the effect of rBGH in the pigs’ carcass compo- sition, transgenic and nontransgenic (control) pigs were raised under the same conditions and fed the same type of diet. The animals were processed at five different live weights: 14, 28, 48, 68, and 92 kg. The entire left side of each carcass was ground, and random samples of tissue were collected and ana- lyzed for fatty acid and cholesterol content. The researchers observed that as live body weight in- creased, carcasses from transgenic pigs showed a constant decline in the amount of total fat compared to control pigs (Table 3.1). Although the results did not demonstrate a difference in the cholesterol con-

tent of transgenic and control pigs, it was shown that transgenic pigs expressing BGH had a significant decrease in the levels of specific fatty acids com- pared with nontransgenic pigs in the control group (Fig. 3.11). These results indicate that consumers might greatly benefit from a pork product with a low fat content if regulation of BGH secretion levels can be precisely controlled during the fast growth stage of young pigs (Solomon et al. 1994).

TRANSGENICPOULTRY: EGG ASBIOREACTOR

Mammals and birds have been the focus of intense research for their possible use as bioreactors. The use of mammals as bioreactors became possible with the creation of transgenic mice and the isola- tion of tissue-specific promoters (Gordon et al. 1980, Swift et al. 1984). Clark et al. (1987) were the first to propose the use of transgenic livestock mam- mary glands for the production of biopharmaceuti- cal proteins in milk. Although expression of foreign protein in milk is high and milk production is large, there are some problems associated with the use of mammary glands as bioreactors, including the long time required to establish a stable line of transgenic founder animals and the high cost to purify foreign protein from milk (Ivarie 2003). Researchers have also long envisioned using chicken eggs for the expression of exogenous proteins. There are many advantages associated with the use of eggs as biore- actors, including the fact that a single ovalbumin gene controls most of the proteins in egg white (Gilbert 1984). Also, egg white has a relatively high protein content, is naturally sterile, and has a long shelf life (Tranter and Board 1982, Harvey et al.

Table 3.1. Comparison of Total Carcass Fat

(g/100g) between rBGH Transgenic and Control Pigs, Measured at Different Live Weights

Total Fat, g/100g Weight Group, kg Transgenic Control

14 6.19 10.04

28 7.62 12.32

48 8.16 16.58

68 5.97 26.78

92 4.49 29.07

2002). There is an already established infrastructure for the production, harvesting, and processing of chicken eggs (Ivarie 2003).

Recently, a group of researchers from the biotech company AviGenics, Athens, Georgia, successfully introduced, expressed, and secreted a bacterial gene in the egg white of transgenic chicken (Harvey et al. 2002). The transgene chosen was the E. coli-lacta- mase (EC 3.5.2.6) reporter gene because it is easily secreted and assayed from eukaryotic cells. A repli- cation-deficient retroviral vector, named NLB, from the avian leucosis virus (ALV) was used to express the transgene. The -lactamase coding sequence was inserted into the pNLB-CMV-BL viral vector and placed under the control of the ubiquitous cyto- megalovirus (CMV) promoter. The protein -lacta- mase was found to be biologically active and was secreted in the blood and egg white, and its expres- sion levels remained constant across four genera- tions of transgenic hens. These results demonstrate

that it is technically possible to express and secret foreign proteins in the chicken egg, making it an attractive candidate for a bioreactor. The main work that needs to be done with the chicken model is to develop more efficient nonviral-based methods for creation of transgenic chicken and to identify, iso- late, and characterize gene enhancers and promoters that have high activity and drive tissue-specific expression of proteins in adult oviducts (Harvey et al. 2002).