Transgenic Cow

Transgenic CowGroup 12:Taca, Carl Angelo V.Dungca, Billy JoelSoliman, DanicaMacasaquit, RosetteTransgenic cows

Scientists at AgResearch in New Zealand have successfully produced healthy transgenic cows that make modified milk or human therapeutic proteins in their milk.

Fur of Transgenic Cow

Transgenic Cow with CalfWhat is a transgenic cow?

Transgeniccows aregenetically modified(GM) cows. They have an extrageneor genes inserted into theirDNA. The extra gene may come from the samespeciesor from a different species.Techniques used to make transgenic cows

Scientists producing transgenic cows use a range of techniques including DNA cloning, restriction enzyme digests, ligation, polymerase chain reaction (PCR), transfection, nuclear transfer and in vitro embryo production.Scientists at AgResearch have successfully generatedtransgeniccows that produce extra proteins in their milk using the 7 steps described below.

Step 1. Designing the gene construct

The first step is to design ageneconstruct. The gene construct is a unit ofDNAthat includes:anantibioticresistancegene to select cells that have taken up the gene constructa tissue-specificpromotersequence to signal the start of expression of theproteinin cells of the appropriate tissue, for example, in mammary cells in lactating cowsthe desired gene for example, bovine casein or human myelin basic proteina stop sequence to define the end of the information for making the protein.

Step 2. Sourcing the transgene

In the past, the gene would have been extracted from the source organisms DNA. Now, however, if the desired gene sequence is known, it can simply be synthesised in a lab. There are companies that make genes to order within a couple of weeks.Step 3. Making the gene construct

The gene is usually supplied in avector. A vector is a small piece of DNA, often aplasmid, into which a foreign piece of DNA can be inserted. When the gene of interest is in a vector, it can be sent from one lab to another, it can be stored, it can be manipulated or it can be used to transformbacteriato produce more copies of the gene of interest.Vectors have multiplerestriction enzymessites (also called multiple cloning sites) so the gene can be inserted into the vector and then cut out from the vector using restriction enzymes.After the gene is cut from the vector, it is then pasted into the multiple cloning site of the gene construct using a method known asligation.

Step 4. Transfecting bovine cells

The gene construct is incorporated into thegenomeof a bovine (cow)cellusing a technique called transfection. During transfection, holes are made in the cellmembranethat allow 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 thenucleusand incorporate into the cells genome.Step 5. Selecting for transgene positive cells

After transfection, an antibiotic is added to select the bovine cells that have incorporated the gene construct. Transgenic bovine cells will survive treatment with an antibiotic, because they contain an antibiotic resistance gene making them resistant to the antibiotic. Cells without the gene construct will have no resistance to the antibiotic and will die. In addition to antibiotic selection,polymerase chain reaction(PCR) is used to check that the bovine cell contains thetransgene.Step 6. Making a transgenic embryo using nuclear transfer

Nuclear transfer is used to create a whole animal from a single transgenic bovine cell.The transgenic bovine cell is fused with a bovine oocyte that has had its chromosomes removed (called anenucleatedoocyte). An electrical pulse is applied to help fuse the cells. Once fused with the oocyte, the transgenic cells chromosomes are reprogrammed to direct development into anembryo. After 7 days, the transgenic embryo will have about 150 cells and can be transferred into arecipientcow for further development to term.

Step 7. Confirming the cow is transgenic

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:PCR to determine the presence or absence of the transgenequantitative PCR (q-PCR) to determine the number of copies of the transgenefluorescent in situ hybridisation (FISH) to visualise where the transgene is in thechromosomeand whether the transgene has integrated into more than one chromosome.When the calf is lactating (either after being induced to lactate or after having its ownprogeny), its milk is checked to determine if the transgenic protein is being expressed

Two Essential Products Produced by Transgenic CowsTransgenic cows making modified milkTransgenic cows making therapeutic proteinsTransgenic cows making modified milk

Cows with modified milk AgResearchs firsttransgeniccows had extra bovine (cow) kappa casein genes inserted in theirgenome. This resulted in increased kappa casein in their milk as the transgenic cow had their 2 naturally occurring kappa casein genes along with the inserted kappa caseingene(s).This research was the first proof that transgenic technology could be used to modify milk composition in cows. Previous research by other groups overseas had focused on using transgenic cows to produce therapeutic proteins.

Casein is a key milk protein

Cows milk is made up of water, sugar, fat, proteins, vitamins and minerals. Casein makes up about 80% of the totalproteinin milk. It is one of the most valuable components of milk because of its nutritional value and processing properties. Casein is particularly important in cheese making.Cows milk is made up of water, sugar, fat, proteins, vitamins and minerals. Casein makes up about 80% of the totalproteinin milk. It is one of the most valuable components of milk because of its nutritional value and processing properties. Casein is particularly important in cheese making.Caseins special structure

Casein proteins come in 4 differentvariants: alpha-s1, alpha-s2, beta and kappa casein. Eachvarianthas its ownamino acidcomposition, genetic variations and functional properties. These 4 casein variants combine to form a structure called amicelle. Micelles are dispersed in the water phase of milk and give milk its white colour .KeyA: submicelleB: protruding chainC: calcium phosphateD: kappa caseinE: phosphate group

Uses of modified milk

benefit animal health, for example, by improving growth and survival of calvesprevent animal diseases, such as mastitismake milk with human health benefitsassist milk processing into dairy products.

Transgenic cows making therapeutic proteins

What are therapeutic proteins?Therapeutic proteins are used to treat human diseases. They include hormones, antibodies, vaccines, growth factors and blood clotting factors.Making therapeutic proteins

Therapeutic proteins are usually synthesised in cell-based systems. In these systems, the desiredgeneis added to acell, and the cells own machinery makes theprotein. The 4 systems currently used to make therapeutic proteins are:microorganisms, for example,yeast,bacteriaor fungiplantsmammalian cellstransgenicanimals.

Advantages making proteins in cows

The main advantages of making therapeutic proteins in transgenic cows are:capacity large amounts of protein are produced the average dairy cow can produce more therapeutic proteins (about 510g/L) than mammalian cells in culture (about 1g/L)cost transgenesis is cheaper than mammalian cell culturescale it is easy to scale production up or down to meet demandfunction proteins are folded and fully functionalharvesting proteins can readily be purified from milk.

Disadvantages of making proteins in cows

The main disadvantages of making therapeutic proteins in cows are:it takes a long time to generate and validate transgenic cowsproteins cant be harvested untillactationbeginsthetransgenemay affect the cow, for example, as the transgene inserts randomly into achromosome, it may disrupt a gene required for an important function.

Transgenic cows on the farm

Transgenic herds live on special farms with their own milking sheds. They are kept separate from regular herds. Transgenic cows look identical to normal cows. Researchers use ear tags and microchips to identify transgenic cows and their calves.One of the aims of the research programme is to show that transgenic cows pass on their transgenes to subsequent generations. If a transgenic cow is mated with a transgenic bull, she will have a higher chance of having transgenic offspring. However, if a transgenic cow is mated with a non-transgenic bull, her offspring will have a 50% chance of being transgenic, as offspring inherit half of their chromosomes from their mother and half from their father.

Uses of transgenic cows

BiomedicineMaking therapeutic proteinsModelling diseaseMaking functional foodsAgricultureImproving food quality or making novel food productsImproving animal healthEnvironmental sustainability

Source:http://www.biotechlearn.org.nz/focus_stories/transgenic_cows