Nuclear Transfer of Embryonic Cell Nuclei to Non-enucleated Eggs in Zebrafish, Danio rerio

Generation of clonal zebrafish will facilitate large-scale genetic screening and help us to overcome other biological and biotechnological challenges due to their isogenecity. However, protocols for the development of clonal lines have not been optimized. Here, we sought to develop a novel method for generation of clonal zebrafish by androgenesis induced by cold shock. Androgenetic zebrafish doubled haploids (DHs) were induced by cold shock of just-fertilized eggs, and the eggs were then heat shocked to double the chromosome set. The yield rate of putative DHs relative to the total number of eggs used was 1.10% ± 0.19%. Microsatellite genotyping of the putative DHs using 30 loci that covered all 25 linkage groups detected no heterozygous loci, confirming the homozygosity of the DHs. Thus, a clonal line was established from sperm of a DH through a second cycle of cold-shock androgenesis and heat-shock chromosome doubling, followed by genetic verification of the isogenic rate confirming the presence of identical DNA fingerprints by using amplified fragment length polymorphism markers. In addition, our data provided important insights into the cytological mechanisms of cold-shock–induced androgenesis.

Section: Resultsmentioning

confidence: 99%

Generation of clonal zebrafish line by androgenesis without egg irradiation

Hou

Fujimoto

Saito

et al. 2015

“…This can be explained by the use of non-enucleated eggs. In medaka 35–38 and in zebrafish cloning 39 , after single blastula nuclei transplant into non-enucleated unfertilized eggs, the NTs that grew to the adult stage expressed genetic markers of both donor and recipient. Following Fatira et al .…”

Section: Discussionmentioning

confidence: 99%

“…This is in accordance with our previous experiment using interspecific single SCNT, in which the obtained NT larva displayed only recipient sterlet genome as a result of unusual disruption in early embryogenesis 11 . Gynogenesis has been observed after fish SCNT when non-enucleated recipient eggs were used: zebrafish intraspecific embryonic cell nuclear transfer resulted in adult diploid NTs (1.6%) that originated only from the recipient 39 . In medaka SCNT, adult NTs (1.0%) exhibited only recipient-derived markers 40 .…”

Section: Discussionmentioning

confidence: 99%

A newly developed cloning technique in sturgeons; an important step towards recovering endangered species

Fatira

Havelka

Labbé

et al. 2019

Several steps of sturgeon somatic cell nuclear transfer (SCNT) have been recently established, but improvements are needed to make it a feasible tool to preserve the natural populations of this group of endangered species. The donor cell position inside the recipient egg seems to be crucial for its reprogramming; therefore by injecting multiple donor somatic cells instead of a single cell with a single manipulation, we increased the potential for embryo development. Using the Russian sturgeon Acipenser gueldenstaedtii as a multiple cell donor and sterlet Acipenser ruthenus as the non-enucleated egg recipient, we obtained higher proportion of eggs developing into embryos than previously reported with single-SCNT. Molecular data showed the production of a specimen (0.8%) contained only the donor genome with no contribution from the recipient, while two specimens (1.6%) showed both recipient and donor genome. These findings are the first report of donor DNA integration into a sturgeon embryo after interspecific cloning. In all, we provide evidence that cloning with the multiple donor somatic cells can be feasible in the future. Despite the fact that the sturgeon cloning faces limitations, to date it is the most promising technique for their preservation.

“…Although enucleated eggs were often used as recipient in the traditional nuclear transplant, non-enucleated eggs have also be applied to reconstruct the SCNT embryos by some researchers owing to its features of simplification and harmlessness in the reconstruction of embryos 22 23 . For example, in medaka, the diploid and fertile reconstructed fish have been successfully generated by transplanting the diploid nuclei into non-enucleated unfertilized eggs 23 24 . Furthermore, it was found that the SCNT fish with the recipient of non-enucleated diploidized eggs only has genetic characteristics of the donor, other than the recipient 25 .…”

Section: Discussionmentioning

confidence: 99%

Autotetraploid cell Line induced by SP600125 from crucian carp and its developmental potentiality

Zhou

Wang

Jiang

et al. 2016

Polyploidy has many advantages over diploidy, such as rapid growth, sterility, and disease resistance, and has been extensively applied in agriculture and aquaculture. Though generation of new polyploids via polyploidization has been achieved in plants by different ways, it is comparatively rare in animals. In this article, by a chemical compound, SP600125, polyploidization is induced in fish cells in vitro, and a stable autotetraploid cell line has been generated from diploid fibroblast cells of crucian carp. As a c-Jun N-terminal kinase (Jnk) inhibitor, SP600125 does not function during the induction process of polyploidization. Instead, the p53 signal pathway might be involved. Using the SP600125-induced tetraploid cells and eggs of crucian carp as the donors and recipients, respectively, nuclear transplantation was conducted such that tetraploid embryos were obtained. It suggests that combining polyploidization and the somatic cell nuclear transfer technique (SCNT) is an efficient way to generate polyploidy, and the presented method in this research for generating the tetraploid fish from diploid fish can provide a useful platform for polyploid breeding.