This interactive shows how scientists confirm a newborn calf is a transgenic animal.
View the steps AgResearch scientists use to produce a transgenic cow in this related interactive.
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.
Acknowledgement: Malaghan Institute of Medical Research
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.
Acknowledgement: Zhang Yongxin, licenced through 123RF Ltd