I find it difficult to split oocytes in medium (hepes buffered T199 + 20% FBS) because the oocytes are usually not stable in the microdrops for efficient splitting during HMC. Is there any better protocol for making the oocytes stable/attached to the base of microdrop during splitting? Thank you all.
Dear Ndubuisi,
you mean really the oocytes? This will not work. You need the embryo, not the oocyte. Hand made cloning works well in the blastocyst stage, where the problems you described are not present.
Pavel Travnik
Dear Pavel,
Thank you for your response. Actually I meant splitting a metaphase 11 oocyte after in vitro maturation. According to Gábor Vajta who developed the technique of hand made cloning, the method involves cutting eggs from sows’ ovaries into two halves under an ordinary microscope. The halves with cell nuclei are discarded, leaving half, empty eggs. A cell (e.g. somatic cells) from the mother sow which is to be cloned is first brought together with one empty half, fused and activated by electrical or chemical method. The eggs are then put into an incubator where they develop into blastocyst, which then are implanted into the sow’s uterus (
Dear Ndubuisi
I usually do HMC in bovine and sometimes I have the same problem but you should probably check the quality of the microblade rather than the oocyte attachment. If the microblade is not new or you have used it for a long time, it will damage the oocyte and it will be more difficult to keep the oocyte “still”. I also increased the FBS concentration to 30% in the manipulation medium. Be sure that the oocyte is in the right position and then try to fix the tip of the microblade to the bottom of the plate and push down firmly over the oocyte. I can send you a video if you need it.
Lleretny Rodriguez-Alvarez
Dear LIeretny,
I warmly welcome and appreciate your comment and suggestions. I was faced with the same challenge of damage to the oocyte as I tried to cut and had to change to a new blade with little improvement. It was difficult to keep the oocytes "still". My next trial will be to raised FBS concentration to 30% and follow your approach as indicated in your comment.
I hereby humbly request a video of the procedure from you as it will be of great assistance to me. ([email protected])
Thank you!
I have experience in both cattle and buffalo cloning, In my view you need to do more practice on these experiments, as HMC is art than science. In early stage difficulty faced for oocytes cutting and will be rectify on by practice. Best wishes_Naresh
Dear Naresh,
I guess you mean 1 or 2ml medium in 35mm dish?
Thanks for the additional information.
Happy New Year 2015!
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