I’m trying to transfer the human chromosomes from A9 mouse hybrid cells (adherent cells) into a lymphoblastoid cell line (suspension cells) but I’m having trouble with seeing any results.
The lab had used the same method before on fibroblast (as recipient) and it worked every time. When I used the LCL as the recipient cells, I was not able to get successful gene transfer. I’m not sure if this is because LCLs are harder to receive chromosomes or if something else was wrong.
Here are my questions:
We are using PEG as a fusion promoting agent and we have been experimenting on the concentration of PEG to add to the microcell solution. Are there any known concentrations that work well for the type of cell lines involved in this fusion process?Right before the fusion step, we are currently adding PHA-P to the microcells and recipient cells and agglutinating for 30 minutes. I wonder if it is sensible to remove the PHA-P after the agglutination process is over (via centrifuge) to increase the concentration of the microcell/recipient cell mixture (which allows the PEG to have maximal contact).We are extracting our microcells through custom-made plates (“bullets”) inserted into centrifuge tubes and we centrifuge these tubes at 27000 x g to extract the microcells. We saw white pellet after centrifugation. However, after we filtered with 8um, 5um and lastly, 3um filter paper to remove the cells, we could not see a pellet (of the micronuclei). Did I lose the micronuclei or it is normal not being able to see the pellet?Another notable difference between the protocol I am following and other existing protocols is that we are using the “bullet” and centrifugation, instead of using a microtubule-disrupting agent, such as colcemid to promote the formation of micronuclei and subsequent microcells and then centrifuging. I am starting to wonder if it’s a better idea to go with the latter process (no bullets), however it’s not something that I have experience with and would like to hear if this is recommended or not.If anyone has experience with any of these, or can reference similar MMCT protocols, any help would be appreciated!
