A preliminary approach for nuclear transfer in goldfish: Reduction in the variability of egg quality, optimization of egg manipulation
The development of nuclear transfer in Goldfish (Carassius auratus) requires egg handling to be optimized 1) to maintain the ability of the egg to withstand embryo development 2) to allow micromanipulation in order to enucleate the egg and inject the donor nucleus 3) to favour egg cytoplasm ability for nucleus reprogramming. In this work, several egg-handling conditions were tested and optimized in the context of nuclear transfer experiments. To obtain the best egg quality, eggs should be left in the female and stripped out just before use, as their ability to withstand embryo development decreased after in vitro storage at 4 or 20°C. Egg enucleation required that the chorion be removed by enzymatic digestion, and a fast method is described which allowed chorion removal within 1 min post activation. After activation, polar body extrusion should be used as an indicator of the pronucleus position underneath, due to failure to label egg DNA. The enucleation success remained low. In experiments where the enucleation proccess was not carried out, chorion removal was therefore not necessary. In this case, egg incubation conditions preventing egg activation were developed in order to improve nucleus reprogramming after injection of eggs with high MPF levels. Trout coelomic fluid proved to be the best inactivation medium when compared to the proteasome inhibitor MG132 and to the soybean trypsin inhibitor. In our experimental conditions, clones were obtained with embryonic and somatic nuclei, but none developed further than the onset of zygotic transcription (1000 cells stage).