Transgenesis is a completely different method farmers use to produce dairy cows with desirable traits. Transgenesis is the process of a gene from one species is transferred into the genome of another species, using a combination of biotechnological techniques. Therefore making proteins that species cannot normally build. Transgenic cows are genetically modified cows. They have an extra gene or genes inserted into their DNA. The extra gene may come from the same species or from a different species. Cows are an attractive target for transgenesis because they naturally secrete large quantities of protein in their milk. This means that, if done correctly, the protein encoded by a transgene will be expressed in the cows milk and can easily be isolated. Thus proteins of therapeutic benefit (to treat human diseases) could be produced in large quantities, relatively inexpensively. Transgenesis are common for the use of developing traits for economic purposes or diseased species. In New Zealand, the AgResearch team have been successful in producing transgenic cows that make modified fatty milk and for therapeutic functions. Transgenic cows have an extra gene present in every cell, expressed only in mammary tissue, thus the transgenes protein is only found and extracted from the cows milk. .
Making a transgenic dairy cow is a process with several elements to it. A variety of techniques in which scientists use to produce transgenic cows includes DNA cloning, restriction enzymes, ligation, polymerase chain reaction (PCR), transformation, nuclear transfer and vitro embryo production.
The first stage of transgenesis is to identify the trait. Scientists analyse the favourable alleles with desirable characteristics that is wanted in the transgenic cow.
Thereafter the gene is extracted from the cows DNA. The scientists obtain the sequence from a genomic library, a collection if cloned segments of DNA containing one copy of at least every gene from the cow. The DNA product contains the cows entire DNA sequence, therefore it is the desired trait as well as the rest of the cows DNA.
Once the gene has been identified and located, the scientists remove the gene sequence from the rest of the DNA. The DNA is cut leaving a several pieces with varying lengths, in which one of them is the gene of interest. The transgene will therefore have the specific variant that is needed because the gene of interest will be with the sticky ends.
After isolated, the transgene is made to modify parts of the gene. The gene construct is a unit of DNA that includes a selectable marker gene, a promoter sequence, the desired gene and a terminal sequence. These are all connected with a ligation enzyme and mixed. The product is incubated in a water bath, then, the scientists use the polymerase chain reaction (PCR). PCR is a technique that allows scientists to copy and multiply a piece of DNA several times. The DNA is heated to 98 degrees in order for it to separate into single strands and polymerase enzyme is added to synthesise new DNA strands from the supplied nucleotides.
Afterwards a technique called transformation will occur in the bovine cells. This is a process where the gene construct is incorporated into the genome of a cow cell. Transformation involves the delivery of a transgene into the nucleus of a recipient cell and integration into a chromosome so it can be passed onto the offspring. Due to cows having a large number of cells, it would impossible to insert a copy of the transgene into every cell, there tissue cultural techniques must be used. Tissue culture is a technique obtaining samples of tissue growing it outside the body without scaffold, and reapplying it a bovine cell line is cultured in an incubator. During the transformation, holes are made in the cell membrane allowing the DNA to enter. The holes can be made in the cell membrane allowing the DNA to enter. The holes can be made by applying an electrical pulse or by adding chemicals to the cells. Once inside the cell, the gene construct may enter the nucleus and incorporate in the cells genome. That can be done either by using an actual stimulus that interferes with the membrane that allows for a short time for the DNA to enter a cell or just by chemical reactions reagents that again interfere with a membrane that surrounds the cell and then allows temporarily for a DNA molecule to enter. The recipient genome is exposed to the transgenes in hopes that a few of the transgenes will actually be integrated into that recipient genome and then properly expressed. This is a rare case therefore there is slight concern that transformation may be indirect after the expression of other genes due to the unpredictable integration of the transgene resulting in a toxic phenotype. Transformation in the bovine cell is necessary because directly injecting transgene into a cow will only change the somatic cells, however the aim is for the gametes to be passed onto the offspring.
To know if the gene has successful incorporated, it needs to screen the cells in order to select the transgene positive cells. The cells are transferred to a selective growth medium containing an antibiotic or chemical, depending on the selectable marker that was used. After the antibiotic or chemical is added, the cells that haven’t taken up the transgene will die. The others will survive because they contain an antibiotic resistance gene making them resistant. The surviving cells will divide and form identical cells. Then it is involved in the PCR to photocopy a whole lot of copies of the gene in order to visualize the transgene present. The two strands in the DNA double helix are separated in denaturation, when the temperature of the DNA solution is raised. The hydrogen bonds between the complementary strands break and the two strands separate. Next the temperature is lowered and an enzyme joins free DNA nucleotides together. The order in which the nucleotides are joined to the new strand is determined by the sequence of nucleotides in the original DNA which is being copied. The result is a double stranded DNA molecule which contains one newly made strand and one original strand. After the newly created double helix is separated, the cycle is repeated.
The cells are tested by a technique which includes gel electrophoresis. It is a tool used to separate and sort fragments of DNA that have ‘cut up’ using restriction enzymes. To perform it, the bovine cell DNA is digested by restriction enzymes and run out on a gel. The DNA fragments are placed into hollows in the gel, then the electric current creates positive and negative ends to a gel plate. DNA will move towards the positive as the phosphate backbone is negative.
Making a transgenic embryo using nuclear transfer and cloning Nuclear transfer is used to create a whole animal from a single transgenic bovine cell. The generation of a transgenic calf follows the same process as the generation of a cloned calf. Ovaries are collected from cows processed at the local abattoir. Eggs are removed from the ovaries and matured overnight in a special media. The nuclear material is then removed from the egg using a fine glass needle and a single cultured cell (carrying the transgene) is positioned against the cytoplasm of the egg (injection). The transgenic bovine cell is fused with a bovine oocyte (egg). An electrical pulse is applied to help fuse the cells. The reconstruct (egg + fused cell) is then chemically activated and placed into culture for development to begin. Once fused with the oocyte, the transgenic cell’s chromosomes are reprogrammed to direct development into an embryo. After 7 days, the transgenic embryo will become a blastocysts and will have about 150 cells, so they can be transferred into a recipient cow for further development to term.
If the embryo develops to full term, after 9 months, the cow will give birth to a calf. To confirm that the calf is transgenic, scientists can check using:
1. Polymerase Chain Reaction (PCR) – PCR can quickly establish whether the transgene is present or absent in the calf’s DNA.
2. Quantitative PCR (q-PCR) – q-PCR is to quantify how many copies of the transgene have been incorporated into the genome of the cell line. The q-PCR machine is a standard PCR but with the incorporation of a fluorescent dye that shows the amplification of the DNA product live on screen as the reaction carries out.
3. Fluorescence in-situ hybridisation (FISH) – FISH is a technique in which include take a biopsy from the animal, grow up cells back into culture, arrest them at metaphase and prepare some slides with those cells. With the slides is possible to probe where the transgene is in the chromosome and visualize if it has integrated into more than one chromosome.
4. Analysing of protein expressed – When cows are two years old they may have their first calf, this way it is stimulated the lactation and milk production. At this point, the milk can be tested to determine whether transgenic proteins, like casein and myelin basic protein are present.
Assuming the transgene has successfully integrated itself into the genome, it will be present in every cell of the animal that develops and will be passed on to following generations through regular sexual reproduction.
The advantages of transgenesis is increased growth rate, improved disease resistance improved food conversion rates, leaner meat, increased muscle mass, improved nutritional quality and improved wool quality. However there are also disadvantages which include; the inserted gene has more than one function, breeding problems, expensive technique, sometimes leads to mutagenesis and functional disorders and low survival rate of transgenic animals.
Impact over genetic biodiversity, health or survival of individuals and populations Improving livestock and animal health Transgenic technologies could be used to improve animal health by increasing resistance to diseases. When technology using molecular biology was developed, it became possible to develop traits in animals in a shorter time and with more precision. In addition, scientists can improve the size of livestock genetically. Transgenesis can allow larger herds with specific traits.
AgResearch’s first transgenic cows had extra bovine kappa casein genes inserted in their genome. This research proved to the scientists that transgenic technologies could be used to alter milk composition in cows. In the future, modified milk from transgenic cows could be used to benefit animal health, for example, by improving growth and survival of calves, prevent animal diseases, such as mastitis, make milk with human health benefits, assist milk processing into dairy products. Overseas milk or meat products from transgenic animals are not allowed to enter the animal or human food supply in New Zealand.
Creating therapeutic proteins
Transgenic cows can be used as ‘biofactories’ to produce human therapeutic proteins. Therapeutic proteins are used to treat human diseases and they include hormones, antibodies, vaccines, growth factors and blood clotting factors.
In June 2006, the first therapeutic protein made in a transgenic animal was approved for use in Europe and the USA. ATryn, a human antithrombin protein, is made in transgenic goats. The protein prevents blood clots in patients who don’t make their own version of this protein. Products such as insulin, growth hormone, and blood anti-clotting factors have already been obtained from the milk of transgenic cows too. Research is also underway to manufacture milk through transgenesis for treatment of debilitating diseases such as phenylketonuria (PKU), hereditary emphysema, and cystic fibrosis. The A. I. Virtanen Institute in Finland produced a calf with a gene that makes the substance that promotes the growth of red cells in humans.
Scientists at AgResearch have generated transgenic cows that produce myelin basic protein (MBP) in their milk. MBP is part of the insulating layer that surrounds nerves. In patients with multiple sclerosis, this insulating layer is gradually destroyed, which prevents the nerves from communicating. Treatment with human MBP may help reduce symptoms of multiple sclerosis.
Impact over ecosystems
In New Zealand, to start a research as the transgenic cows by AgResearch, it is needed to follow strict guidelines for care and containment of the animals. Transgenic cows are classed as new organisms and are regulated by the Hazardous Substances and New Organisms (HSNO) Act. The HSNO Act is overseen by the Environmental Protection Agency (EPA). The EPA provides rules and regulations for introducing any hazardous substances or new organisms to New Zealand. Before any research can be done, an application must be made to the EPA. EPA evaluates the benefits and risks of any research and decides whether the work can begin. Anyone can make a submission on an application, which can support it, oppose it or support some parts and oppose others. Applications to EPA can be viewed on the EPA website.