In agriculture there has always been a goal to improving the quality and levels of production with milk and milk products. In the past this improvement has been achieved by selective breeding, mainly to increase milk yield. Animals with high production rate were chosen to be used in the breeding of the next generation. This selection was repeated every year each time choosing the best animals available in hope that these traits are passed on to offspring. This later moved on to improvement by using genetic information to increase favourable traits in a population. Genetic information provided a more precise way to measure favourable traits and the possibility that traits are passed on. However, there is a limit to improvements in the conventional way, as there is a low hereditability traits associated with milk production (Pintado et al 1999) and the fact that genes cannot be transmitted from one species to another (Magnus, Lali 2008). Transgenesis is a way to introduce new genetic traits into animals that otherwise cannot be introduced. A transgenic animal is one which has had a new gene added to their genome from another species. Transgenic animals are used to improve the quality of the products they produce (milk/meat) for the use of humans. The process of adding these genes into animal genomes to produce new protein in animals is called biopharming. Biopharming has a number of uses, from adding genes to milking animals producing biopharmaceuticals (proteins used for therapeutic reasons added form another biology source) in their milk to genetically manipulating genome of animals for such project as xenotransplants. In this the main focus will be on the use of transgenesis to produce transgenic milk for the use in biopharmacology. Transgenic milk is the product of genetic engineering by transferring a gene which contains a protein that is not present in the animal into their genome, this then leads to the protein being expressed in mammary gland of the transgenic animal, which is in turn expressed in their milk.
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HISTORY OF GENETICS: FROM NATURAL SELECTION TO TRANSGENESIS
The evolution of genetics and the science around it has moved at a rapid pace. Genetics was first described in 1858 by Charles Darwin with his theory of natural selection. This was published a year later 1859 by Darwin in his book The Origins Of Species. This was followed in 1866 by Gregor Mendel who first described genetic traits and their relationship to each other in his experiments on pea plants. In 1910 Thomas H Morgan begins study on the Drosophila fly which became very important species in understanding genetics. This became the model for developmental biology. In was in 1953 when Francis Crick and James Watson first produced a 3D model of DNA enhancing the understanding of DNA. In 1970 Hamilton SmithÂ and Kent Wilcox first isolated the restriction enzyme, HindII, which made cutting DNA at specific recognition sites possible. It was in 1972 that Paul BergÂ and Herb Boyer produced the first recombinant DNA molecule. Kary B. Mullis in 1985 first described PCR, a breakthrough in genetics. In the 1990s whole genome sequences begin for humans. This all led up to cloning of animals and finally the invention of transgenic animals and transgenic produces. (Lane 1994)
The process of transgenic can be done in two ways, pro nuclear microinjection or nuclear transfer technique. Transgenesis is a process which involves the injection of foreign DNA into a nucleus of an embryo. The foreign DNA consist a region that has a gene (transgene) which encodes a protein of interest (Keefer et al 2004). This protein that is expresses is called the recombinant protein.
Pronclear microinjection involves the adding of a new gene to an embryo cell and implanting the embryo in a surrogate mother. Many copies of the targeted gene are injected into the pronuclei of a fertilized oocyte in hopes that the transgene is incorporated in the DNA and results in a new transgenic animal (Keefer 2004). However there is limitation to this process. The gene is not always incorporated fully and can result in animals with only some cells that have the transgene (Keefer 2004). Only a small proportion of embryos injected will become transgenic, integration of the transgene into the chromosomes are random and cannot be controlled (Colman 1999). There is also no guarantee that the transgenic animal will express enough of the protein to be considered economic. There is also the problem that less than 50% of offspring of transgenic animals will carry the transgene.
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There are limitations to the microinjection technique as the process is slow and the ability to generate a founder animal is often hard. Nuclear transfer is more efficient, this is a technique which involves the transfer of the nucleus DNA of a somatic cell into an enucleated oocyte. In this process the new oocyte is reactivated to the embryonic developmental stage (Keefer 2004). The advantage of this method over microinjection technique is that the donor cell can be screened for the transgene prior to insemination ensuring the produced animal is transgenic (Keefer 2004). This would lead to a new individual that is identical to the donor cell (Tain et al 2003). A donor cell is collected from an adult. Another egg cell is removed from a different adult animal, the nucleus is removed. The donor cell and the enucleated are fused together. The new cell is then cultured to form an embryo. This embryo is then transferred into a surrogate mother. When the offspring is born they have the exact copy of the DNA as the donor. This is illustrated by figure 2 below. Somatic cell are used during this process, they are used to generate multiple copies of animal either to create a new animal of high genetic merit (same as the donor) or to be manipulated further to produce transgenic animals to produce pharmaceutical proteins (Tain et al 2003). This manipulation is done by injecting the somatic cell's DNA with a new gene, similar to microinjection.
THE USE OF BIOPHARMACEUTICALS IN TRANSGENIC MILK
There needs to be a reason why a gene of interest is chosen, it is normally based on economically, scientific and social reasons (Keefer 2004). An important part of selecting the gene in question is if and when this recombinant gene is expressed what is the purpose of it, improvement of a product, find a new treatment for a disease or just for scientific research (Keefer 2004). Another important point to take into account when dealing with selection of genes weather the transgene will affect the animal's natural physiology. In other word if the foreign DNA will have a negative effect on the health on the transgenic animal (Keefer 2004).
Today milk is one of the most important food source, used both in its natural form as well as milk products such as chesses and yogurts. Increasing milk proteins is the best way to improve the quality of milk and milk products (Brophy et al 2003). An important application of the process of transgenics is to produce animal products with biopharmaceutical in them. There are four categories in which the production of transgenic milk fall into
Adding value to products
Improving nutritional value
Production of proteins for health
Production of biomaterials (Keefer 2004)
The first livestock had a gene inserted with expressed the human growth hormone which made animals grow faster (Whitelaw 2004). This led to many experiences in transgenesis of other genes. Through transgenics it is possible to produces proteins for human consumption in the mammary cell of animal and in their milk. This alters the composition of milk to increase proteins and other nutrient. This has an intense effect on the growth rate and survival of offspring, and in the human who drink the transgenic milk (Wheeler et al 2003). It is an important part of transgenesis that transgenic animal are created which can be used for the production of proteins. It is important that the transgene is expressed in the mammary cells and can be transferred to the milk during lactation (Brink et al 2000).
Another major use in transgenic milk would be the effect on health, animals or humans. With transgenics it is possible to produce milk that contains antibodies, which can reduce the risk of mastitis and infections. There is also the possibility of producing antibodies that can fight animal and human diseases (Wheeler et al 2003). The hope is to produce "medicine milk" which has the ability to have antibodies present as a form of treatment (Magnus, Lali 2008). The expression of proteins in transgenic milk of livestock is a major advantage of commercial application of transgenic animals (Salamone et al 2006). The development of effective application to exploitation of this technology is needed (Pollock et al 1990). Human products such as insulin, growth hormones and anti-clotting factors have been produced in milk of transgenic animals, sheep goats and cows (Magnus, Lali 2008). It is important to produce large numbers of recombinant therapeutic proteins to supply the demand needed for trials with large numbers of patients where large amounts of the proteins are needed (Pollock et al 1999).
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The overall reason to produce transgenic milk is ultimately to improve the quality of milk and milk products and to uses these products in the medical and health industry (Wheeler et al 2003). However the process of transgenesis is highly inefficient (Whitelaw 2004) and there is a need to improve the efficiencies of the process and so it reduces the cost.
Biopharmaceuticals available in milk
There has been many project and experiments in producing biopharmaceuticals in milk. These experiments have been carried out on many animals form mice to large domestic animals such as cattle. The choice of animal depends on the amount of the interested protein that is needed (Pollock et al 1999). These proteins range from improving the quality of milk by introducing ceasin protein in cattle to one of the first proteins added alpha 1 antitrypsin in sheep. The major research projects focus on the three major sources of milk cows, sheep and goats. The used of transgenic mice is very usefully in testing if transgenes can be expressed and if the express of the gene can harm the transgenic animal. They can't be used for produce as the quantity of milk produced is insufficient (Pollock et al 1999). Projects using the smaller dairy animals (sheep and goats) cost less than the dairy animal (cows) (Keefer 2004). The below table shows some of the transgenic animals to date.