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1.
Mar Biotechnol (NY) ; 20(3): 324-342, 2018 Jun.
Article in English | MEDLINE | ID: mdl-29679251

ABSTRACT

Repressible knockdown approaches were investigated to manipulate for transgenic sterilization in channel catfish, Ictalurus punctatus. Two primordial germ cell (PGC) marker genes, nanos and dead end, were targeted for knockdown and an off-target gene, vasa, was monitored. Two potentially copper-sensitive repressible promoters, yeast ctr3 (M) and ctr3-reduced (Mctr), were coupled with four knockdown strategies separately including: ds-sh RNA targeting the 5' end (N1) or 3' end (N2) of channel catfish nanos, full-length cDNA sequence of channel catfish nanos for overexpression (cDNA), and ds-sh RNA-targeting channel catfish dead end (DND). Each construct had an untreated group and treated group with copper sulfate as the repressor compound. Spawning rates of full-sibling P1 fish exposed or not exposed to the constructs as treated and untreated embryos were 85 and 54%, respectively, indicating potential sterilization of fish and repression of the constructs. In F1 fish, mRNA expressions of PGC marker genes for most constructs were downregulated in the untreated group and the knockdown was repressed in the treated group. Gonad development in transgenic, untreated F1 channel catfish was reduced compared to non-transgenic fish for MctrN2, MN1, MN2, and MDND. For 3-year-old adults, gonad size in the transgenic untreated group was 93.4% smaller than the non-transgenic group for females and 92.3% for males. However, mean body weight of transgenic females (781.8 g) and males (883.8 g) was smaller than of non-transgenic counterparts (984.2 and 1254.3 g) at 3 years of age, a 25.8 and 41.9% difference for females and males, respectively. The results indicate that repressible transgenic sterilization is feasible for reproductive control of fish, but negative pleiotropic effects can result.


Subject(s)
Animals, Genetically Modified/metabolism , Embryo, Nonmammalian/metabolism , Germ Cells/metabolism , Ictaluridae/metabolism , Animals , Animals, Genetically Modified/genetics , Copper/metabolism , Ictaluridae/genetics , Polymerase Chain Reaction , RNA Interference , Reproduction/genetics , Reproduction/physiology , Sterilization, Reproductive/methods
2.
Sci Rep ; 7(1): 7301, 2017 08 04.
Article in English | MEDLINE | ID: mdl-28779173

ABSTRACT

The myostatin (MSTN) gene is important because of its role in regulation of skeletal muscle growth in all vertebrates. In this study, CRISPR/Cas9 was utilized to successfully target the channel catfish, Ictalurus punctatus, muscle suppressor gene MSTN. CRISPR/Cas9 induced high rates (88-100%) of mutagenesis in the target protein-encoding sites of MSTN. MSTN-edited fry had more muscle cells (p < 0.001) than controls, and the mean body weight of gene-edited fry increased by 29.7%. The nucleic acid alignment of the mutated sequences against the wild-type sequence revealed multiple insertions and deletions. These results demonstrate that CRISPR/Cas9 is a highly efficient tool for editing the channel catfish genome, and opens ways for facilitating channel catfish genetic enhancement and functional genomics. This approach may produce growth-enhanced channel catfish and increase productivity.


Subject(s)
CRISPR-Cas Systems , Gene Editing , Ictaluridae/genetics , Myostatin/genetics , Zygote/metabolism , Animals , DNA Mutational Analysis , Female , Mutagenesis , RNA, Guide, Kinetoplastida
3.
Mar Drugs ; 15(6)2017 May 31.
Article in English | MEDLINE | ID: mdl-28561774

ABSTRACT

Repressible knockdown approaches were investigated for transgenic sterilization in channel catfish, Ictalurus punctatus. Two primordial germ cell (PGC) marker genes, nanos and dead end, were targeted for knockdown, and an off-target gene, vasa, was monitored. Two potentially salt sensitive repressible promoters, zebrafish adenylosuccinate synthase 2 (ADSS) and zebrafish racemase (Rm), were each coupled with four knockdown strategies: ds-sh RNA targeting the 5' end (N1) or 3' end (N2) of channel catfish nanos, full-length cDNA sequence of channel catfish nanos for overexpression (cDNA) and ds-sh RNA targeting channel catfish dead end (DND). Each construct had an untreated group and treated group with sodium chloride as the repressor compound. Spawning rates of full-sibling P1 fish exposed or not exposed to the constructs as treated and untreated embryos were 93% and 59%, respectively, indicating potential sterilization of fish and repression of the constructs. Although the mRNA expression data of PGC marker genes were inconsistent in P1 fish, most F1 individuals were able to downregulate the target genes in untreated groups and repress the knockdown process in treated groups. The results indicate that repressible transgenic sterilization is feasible for reproductive control of fish, but more data from F2 or F3 are needed for evaluation.


Subject(s)
Animals, Genetically Modified/genetics , Catfishes/genetics , Germ Cells/metabolism , Ictaluridae/genetics , Reproduction/genetics , Sodium Chloride/metabolism , Animals , Animals, Genetically Modified/metabolism , Base Sequence , Catfishes/metabolism , DNA, Complementary/genetics , Embryo, Nonmammalian/metabolism , Female , Gene Knockdown Techniques , Male , Promoter Regions, Genetic/genetics , Sterilization/methods , Zebrafish/genetics , Zebrafish/metabolism
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