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Making a transgenic animal

An interactive showing the techniques used to make transgenic animals at AgResearch in New Zealand.

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1. Designing the gene construct

To design a gene construct we look at what the overall function will be and we then align it all logically. It will require the promoter, the gene of interest, and then a poly A tail. Because we need to amplify up or make more copies of this gene so that we have bulk to work with we have bacterial sequences involved so that it can be amplified within bacteria.

For most of our applications we are designing our construct that they are only active in their mammary gland, so the expression is only at late pregnancy and during lactation, the then protein produced in the mammary gland will then be secreted into the milk.

2. Sourcing the transgene

The process in getting the particular transgene we want depends on the transgene, so right now we are doing work for an external company, and they are providing us with that transgene. If its it’s a transgene that can't be provided by an external source we will then have that synthetically made ourselves, either in our lab or we will go to an external manufacturer.

So if we are looking for a transgene that specifically expresses myelin or the casein we would ask for that gene and the variant that we are interested in.

It would probably come within a vector with restriction enzyme sites on either side of it that we can just use restriction enzymes to cut it out, and in that process it would leave our gene of interest with what we call sticky ends on either side that it can then be easily glued back into a vector that we want to use.

3. Making the gene construct

So what we are doing with ligation is just sticking the gene of interest into an expression vector.

With this example of ligation I have a vector backbone, which is already been opened by a restriction enzyme and has what we call a sticky end. And then I have my gene of interest, which is a short fragment, which also has sticky ends. And then using a ligation enzyme and mixing these two fragments together the sticky ends will be like stuck together that is what a ligation enzyme will do. So that is incubated in the water bath at 16 degrees for half an hour.

4. Transfecting bovine cells

Transfection is the name for the introduction of DNA into a cell. And that can be done either by using an actual stimulus that interferes with the membrane and 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.

We use a delivery package to deliver the DNA to the cells.

So here I have some cells growing in the dish, and one of the methods that we use which is using liposomes, and what liposomes do, is they help package the DNA so that it can be presented to the cells, and that it can easily enter the cell membrane. So what I have here is just some DNA and some liposomes and I'll mix them together and then drop them onto the plate.

In our case to transform bovine cells with a transgene, we have bovine cells growing in culture, and we choose to work with the female line so that at the end we end up with female calves.

5. Selecting for transgene positive cells

To know our gene has successfully incorporated we’ll need to screen our cells; that our transgene will have an antibiotic resistant gene as well. So We then apply antibiotics to our cells and the cells that haven’t taken up the transgene will die. And those that have taken up the transgene will continue to survive and they will also divide and form a small colony of identical cells, and that is what we are looking for. We do characterise our clones further and that involves some PCR, Polymerase Chain Reaction, it’s an enzymatic reaction which is like using a photocopier and runs off a whole lot of copies of our gene and so that we can run that on a gel and visualize it -that the transgene is actually present. Because sometimes we will have false positives that are growing, but they actually don't have the transgene so we want to look for those.

And then from there once we have confirmed that our cells are transgenic we will hand them onto the cloning team.

6. Making a transgenic embryo using Nuclear transfer

As a broad overview the cloning team have sourced eggs from say the abattoir, and these eggs have had the DNA removed and then our cell, our transgenic cell is fused with this egg so that the DNA of our cell now becomes the DNA of the egg. And that egg is stimulated to grow and cultured in the lab for approximately 7 days and then is transferred into a recipient cow. And we monitor that gestation or the pregnancy over 9 months, and hopefully we have a beautiful calf on the ground at the end.

7. Confirming the cow is transgenic

So once we have the transgene transgenic animals on the ground we do further characterise them. First we go back to basic PCR just to confirm the transgene is there. We are also interested in knowing how many copies of the transgene are present, where within the genome the transgene has incorporated, whether it’s into one position or multiple. As for looking at protein levels, we definitely look at that, the levels of protein expressed within the milk.

7A. Polymerase chain reaction (PCR)

To establish whether a newborn calf is a transgenic animal, scientists use PCR. PCR can quickly establish whether the transgene is present or absent in the calf’s DNA.

7B. Quantitative PCR (q-PCR)

What we can use real- time PCR for is to quantify how many copies of our transgene have been incorporated into the genome of our cell line. And real- time PCR is a great tool for that.

The real- time PCR machine is a standard PCR but with the incorporation of a dye, a fluorescent dye so that we can see the amplification of the DNA product live on screen as the reaction carries out.

So we can run a standard curve where we know how many copies are in our standard curve and compare that back to our transgene or our cell line and work out from there how many copies of the gene is incorporated.

7C. Fluorescence in-situ hybridisation

When looking at how many copies are incorporated and where within the genome we use a technique called FISH which is fluorescence in-situ hybridisation, and I will take a biopsy from the animal and grow up cells back into culture and then arrest them at metaphase, prepare some slides with those cells, and then I will probe my slides for the transgene. And on the slides I will have an array of the chromosomes, and where the transgene has incorporated will glow on those chromosomes. So if there is one site of insertion we will see one site where the probe will glow.

7D. Analysing of protein expressed

When cows are two years old they may have their first calf, this stimulates 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.


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