The transgene is inserted into a gene construct and then the construct is introduced into female bovine (cow) cells. Bovine cells that have successfully incorporated the gene construct survive treatment with antibiotics as the construct contains a gene that expresses resistance to the antibiotic. These cells are then characterised using polymerase chain reaction (PCR) to check that the transgene is present. The next step for these transgenic bovine cells is to be used to produce a transgenic embryo through nuclear transfer.
For further information, see the video: Nuclear transfer.
Jargon alert – Listen out for the words ‘electroporation’ and ‘nucleofection’, which describe specific techniques for introducing foreign DNA into a cell nucleus. Also, listen for the term ‘screening our cells’, which is a process to find the cells that have successfully incorporated the foreign DNA (the transgene) into their genome
Transcript
DR GOETZ LAIBLE
To go from the gene construct to introduce it into the bovine genome, we are using a cell-based method. We are introducing the gene construct into the bovine cell, and not only introducing into the cell, but we are integrating it into the genome.
MARION WOODCOCK
In our case, to transform bovine cells with the 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. And so we've got the cells growing in culture, and once they are at the desired confluency – that means that there is enough cells within the well but not too many – we use a delivery package to deliver the DNA to the cells, so that might be electroporation or nucleofection, and that creates an access into the cells, so the DNA can go into the cell and then deliver the transgene into the nucleus and hopefully then also incorporate it into the genome.
To know that the gene has successfully incorporated, we’ll need to screen our cells that our transgene will have an antibiotic resistance gene as well. So we then apply antibiotic 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’s what we are looking for.
We do characterise our clones further. We do a PCR screen, which is a 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 so that we can run that on a gel and visualise 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. And from there, once we have confirmed that our cells are transgenic, we will hand them onto the cloning team.
