The influence of donor nucleus source on the outcome of zebrafish somatic cell nuclear transfer

Abstract

The success of nuclear reprogramming following somatic cell nuclear transfer (SCNT) is thought to depend on factors present in the egg. Little is known about the role -if any -played by the somatic cell type on the outcome of the procedure. We tested whether cells of different lineages might have different capacities for reprogramming following SCNT, comparing cells isolated from five different tissues of transgenic zebrafish for their developmental potential when used as SCNT donor cells. We used transgenic zebrafish lines expressing green fluorescence protein under an endogenous tissue-specific promoter: HGn62A-skin, HGn28A-skin, HGn8E-heart, HG21C-fin and notochord and HGn30A-hatch gland. We analyzed the efficiency of cloning, as measured by reconstructed embryos that developed up to the hatched-fry stage. Specifically, donor cells of fin and notochord origin yielded the best rate of cloned fish production. All of the other cell types used were capable of producing cloned fish, albeit with significantly lower efficiency. These results indicate that the type of zebrafish cells used for SCNT can influence the outcome of the procedure. Future epigenetic analysis of these cells will help determine specific chromatin profiles in somatic cells that have an impact on nuclear reprogramming procedures.

Application of interspecific Somatic Cell Nuclear Transfer (iSCNT) in sturgeons and an unexpectedly produced gynogenetic sterlet with homozygous quadruple haploid

“…It was striking to observe that all sturgeon NT-embryos that showed initial cleavage furrows continued development through the mid-blastula stage, and that many of them developed up to the gastrula stage. This result is in contrast with previous studies showing that a high percentage of the NT face difficulties to overcome the mid-blastula stage 22 – 24 , 28 , 38 . The later has been reported to be the stage at which the mitotic checkpoints are established 39 , and at which the embryonic genome is activated 40 .…”

A 13-plex of tetra- and penta-STRs to identify zebrafish

“…To this end, single zebrafish embryos were manually dechorionated, euthanized using 0.02% MS222 and bleached in a cold 0.005% solution of bleach in PBS for 5 minutes. Single embryos were then rinsed in PBS three times and transferred to a drop of DNAC medium made of 7% heat-inactivated fetal bovine serum (FBS, Thermo Fisher Scientific, MA), 2% trout serum (Seagrow, EastCoast Bio Inc, CA), 2 mM N-acetyl-L-cysteine (Sigma-Aldrich, MO), 0.2 mM L-ascorbic acid 2-phosphate (Sigma-Aldrich, MO), 50 IU of bovine insulin (Cell Applications Inc, CA), 20 ng/ ml of human insulin growth factor (IGF, PeproTech US, NJ)), and 1% 100X Anti-Anti (Thermo Fisher Scientific, MA) in DMEM (Thermo Fisher Scientific, MA) 8 . With the help of 27-gauge needles, single embryos were cut into small tissue pieces that were subsequently placed in Matrigel-coated wells of a 96-well plate containing 50 µl of DNAC medium.…”

Cryobanking of aquatic species

“…This is especially important in the case of endangered fish or unique specimen, where no drastic injury should impair the precious individual. Besides, fin cells are among the best donor cells for fish regeneration by nuclear transfer (Siripattarapravat et al, 2011). Collection of somatic cells from developing embryos can be trickier as it will require chorion removal (by enzymatic digestion or mechanical shearing) and that after collection of few cells, the embryo is incubated into media with specific ion composition before reaching the hatching stage, in order to cope with the loss of the chorion barrier.…”