Making transgenic bovine cells
The is inserted into a 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 to the . These cells are then characterised using (PCR) to check that the transgene is present. The next step for these bovine cells is to be used to produce a transgenic through nuclear transfer.
For further information, see the video:
Jargon alert – Listen out for the words ‘electroporation’ and ‘nucleofection’, which describe specific techniques for introducing foreign into a . 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
Transcript
DR GOETZ LAIBLE To go from the construct to introduce it into the bovine , we are using a cell-based method. We are introducing the gene construct into the bovine , 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 , we have bovine cells growing in , 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 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 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 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 . 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 , we will hand them onto the cloning team.
