Hi everyone,
I am new to mES field and my task is to establish mES cells from transgenic mice. I have already done it for C57Bl/6 mice but when I switch to C57Bl/6 transgenic mice I get some strange things happen I can't expain: I plate 3,5 day blastocysts on inactivated feeder, change media every other day and after 1 week I try to pick the colony and dissociate it to numerous clumps and to reseed the cells. I can't disaggregate the colony, I wash it in PBS and use 0,25% of trypsine. The colony stays intact even if I pipette it well. Any suggestions please?
Is your trypsin in good activity? Start with a new opened trypsin. Wash cells with PBS two or three times, allow the cells incubated with trypsin long enough until colonies detached. Then and complete medium to stop trypsin digestion and transfer to a 50ml centrifuge tube. Pipette vigorously. Give them more power. ES cells always connect with each other tightly.
If you can do this side by side WT vs transgenic, using all the same methods and materials, and the result stays the same, then you have a phenotype. Is your gene expressed in embryonic stem cells? Can it be expected to have an effect on cell adhesion? Maybe your lab gets to study how it all works, publish papers and win grants :)
Other dissociation methods to look at (typically used for human ESC, which can't be exposed to trypsin and like to be passaged as multi-cell clumps): Accutase, shearing with a narrow syringe needle rather than pipet tip, mechanical cutting using things like "stem cell knife", and ROCK inhibitor.
Hi Elena,
I think John has given you a good advice of doing your WT and transgenic derivations parallel to each other and then see if one is better than the other. I've had times where there would be easy derivation at certain times, and then nothing for a couple of weeks! It could be frustrating, but hang in there. So when I had such problems, (I'm sure you would have tried already, but just if you haven't..) I tried playing around with the trypsin incubation times- keep for 5 minutes, take the plate out, under a fume hood- try to dissociate the picked embryos (in round 96-well plates) under the microscope, then put back for a couple of minutes-say 3-4 more minutes and dissociate again. I had also changed my trypsin concentration. I used to dilute 0.25% trypsin with PBS (1:1) (50ul in 96-well plate) and I also changed the micropipette size, earlier I used to use 200ul, and then I switched to 10ul, fixed at 3-4 ul to break down the embryo.. in Some of my IVF embryos, I often had to be more vigorous in pipetting as most used to have zona pellucida still present around them..
these are just a few tricks I tried, which worked for me (most of the times).. but if you do exact same method 'at the same time'- as John suggested, and still it doesn't work, then maybe the 3.5E blastocyst are somehow different than WT's and might require completely different protocol (which is still hard to imagine.. but its Science- you would never know till you try! ;) )
Hope it helps.
Good luck!
Pooja.
Hi guys,
Thank you for help. In fact I don't know why my transgenic blastocysts look different from WT already at the stage of blastocysts flushing - they don't really look nice as for example I have seen from CD1 mice or BL6 wt. I will perform more tries and I will let you know even if I am kind of desperate. I will order some wt mice and run the experiment in parallel.
Have a nice day!
Hello
Agree with the researchers, has given you a good suggestions
Pleas look at papers here , i hope help you
Good Luck in youer experment
http://www.ncbi.nlm.nih.gov/pubmed/17185608
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2706045/
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