<p>Representative channel catfish male-biased genes involved in spermatogenesis, gonad sex ... more <p>Representative channel catfish male-biased genes involved in spermatogenesis, gonad sex determination, and testicular determination.</p
<p>Note that the proportion of male-biased genes per linkage group is similar except for ch... more <p>Note that the proportion of male-biased genes per linkage group is similar except for chromosomes 1, 6, 8, 7, 24 and 27 (shown in red for linkage groups with high numbers of male-biased genes and in yellow for linkage groups with low numbers of male-biased genes).</p
<p>GO annotations were based on zebrafish RefSeq and GO-terms were processed by Blast2Go an... more <p>GO annotations were based on zebrafish RefSeq and GO-terms were processed by Blast2Go and categorized at level 2 into three major functional categories (biological process, cellular component, and molecular function).</p
Stem cells are a class of undifferentiated cells, have the potential for self-renewal that can di... more Stem cells are a class of undifferentiated cells, have the potential for self-renewal that can differ to the specialized cells. First studies on stem cells in fish started with zebra fish in 1992. In this review, classification, resources, vital importance, characteristics and application areas of fish stem cell were clarified.
Abstract Constructs bearing growth hormone (GH) cDNA from channel catfish, Ictalurus punctatus, d... more Abstract Constructs bearing growth hormone (GH) cDNA from channel catfish, Ictalurus punctatus, driven by the ocean pout, Zoarces americanus, antifreeze protein promoter (opAFP), or by the rainbow trout, Oncorhynchus mykiss, metallothionein promoter (rtMT) were transferred to channel catfish via electroporation. Transgenic P1 individuals were mated to produce F1 individuals that exhibited enhanced growth rate. The inheritance of the transgene by the F1 generation was 10% to 36%. The mean body sizes of F1 transgenic fish were larger than their non-transgenic full-siblings (P 0.05) between transgenic and non-transgenic full-siblings except for one opAFP-ccGH family, although the observed means were smaller for four of six transgenic groups as apparently their relative length was increasing more rapidly than relative weight compared to their respective controls. Examining the data from the current experiment and that from an earlier experiment with the same genetic types, but grown to a smaller size, it appears that the two promoters gave similar results in regards to growth enhancement. The largest individuals were always in the transgenic families, and this along with the family effects suggest that gene transfer coupled with combined individual and family selection might result in larger fish than the GH transgenesis alone. The growth enhancement in the current experiment was better than previous experiments with channel catfish that used RSV-LTR-salmonid GH constructs. Assuming government approval and the same growth to food size, GH transgenic channel catfish would increase farm income by $3421/ha compared to use of non-transgenic channel catfish.
Our aim was to transplant blue catfish germ line stem cells into blastulae of triploid channel ca... more Our aim was to transplant blue catfish germ line stem cells into blastulae of triploid channel catfish embryos to produce interspecific xenogenic catfish. The morphological structure of the gonads of blue catfish (Ictalurus furcatus) in ~ 90- to 100-day-old juveniles, two-year-old juveniles, and mature adults was studied histologically. Both oogonia (12-15 μm, diameter with distinct nucleus 7-8 μm diameter) and spermatogonia (12-15 μm, with distinct nucleus 6-7.5 μm diameter) were found in all ages of fish. The percentage of germ line stem cells was higher in younger blue catfish of both sexes. After the testicular tissue was trypsinized, a discontinuous density gradient centrifugation was performed using 70, 45, and 35% Percoll to enrich the percentage of spermatogonial stem cells (SSCs). Four distinct cell bands were generated after the centrifugation. It was estimated that 50% of the total cells in the top band were type A spermatogonia (diameter 12-15 μm) and type B spermatogoni...
Marine biotechnology (New York, N.Y.), Jan 20, 2018
Repressible knockdown approaches were investigated to manipulate for transgenic sterilization in ... more 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 P 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 F fish, mRNA expr...
Repressible knockdown approaches were investigated for transgenic sterilization in channel catfis... more 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 mR...
Marine biotechnology (New York, N.Y.), Jan 4, 2016
Channel catfish (Ictalurus punctatus) is the most important freshwater aquaculture species in the... more Channel catfish (Ictalurus punctatus) is the most important freshwater aquaculture species in the USA. Genetically enhanced fish that are sterile could both profit the catfish industry and reduce potential environmental and ecological risks. As the first step to generate sterile channel catfish, three sets of zinc finger nuclease (ZFN) plasmids targeting the luteinizing hormone (LH) gene were designed and electroporated into one-cell embryos, different concentrations were introduced, and the Cel-I assay was conducted to detect mutations. Channel catfish carrying the mutated LH gene were sterile, as confirmed by DNA sequencing and mating experiments. The overall mutation rate was 19.7 % for 66 channel catfish, and the best treatment was ZFN set 1 at the concentration 25 μg/ml. To our knowledge, this is the first instance of gene editing of fish via plasmid introduction instead of mRNA microinjection. The introduction of the ZFN plasmids may have reduced mosaicism, as mutated individu...
Testicular germ cells of channel catfish, Ictalurus punctatus, and blue catfish, I. furcatus were... more Testicular germ cells of channel catfish, Ictalurus punctatus, and blue catfish, I. furcatus were separated into four layers with Percoll density gradient centrifugation, containing different cell types (40 % in the first layer were spermatogonial stem cells, SSCs). Expression of seventeen genes was analyzed for cells from different layers by real-time quantitative PCR. Pfkfb4, Urod, Plzf, Integrin6, IntegrinV, Thy1 and Cdh1 genes showed the same expression change pattern in both channel and blue catfish as these genes were down-regulated in the spermatocytes and even more so in spermatids. Plzf and Integrin6 had especially high expression in SSCs and can be used as SSCs specific markers. Sox2 gene was up-regulated in spermatocytes and even more highly up-regulated in spermatids, which indicated it could be a spermatid marker. In contrast to channel catfish, Id4, Smad5 and Prdm14 gene expressions were strongly down-regulated in spermatocyte cells, but up-regulated in spermatid cells...
Channel catfish (Ictalurus punctatus) embryos were electroporated with sterilization constructs t... more Channel catfish (Ictalurus punctatus) embryos were electroporated with sterilization constructs targeting primordial germ cell proteins or with buffer. Some embryos then were treated with repressor compounds, cadmium chloride, copper sulfate, sodium chloride or doxycycline, to prevent expression of the transgene constructs. Promoters included channel catfish nanos and vasa, salmon transferrin (TF), modified yeast Saccharomyces cerevisiae copper transport protein (MCTR) and zebrafish racemase (RM). Knock-down systems were the Tet-off (nanos and vasa constructs), MCTR, RM and TF systems. Knock-down genes included shRNAi targeting 5&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39; nanos (N1), 3&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39; nanos (N2) or dead end (DND), or double-stranded nanos RNA (dsRNA) for overexpression of nanos mRNA. These constructs previously were demonstrated to knock down nanos, vasa and dead end, with the repressors having variable success. Exogenous DNA affected percentage hatch (% hatch), as all 14 constructs, except for the TF dsRNA, TF N1 (T), RM DND (C), vasa DND (C), vasa N1 (C) and vasa N2 (C), had lower % hatch than the control electroporated with buffer. The MCTR and RM DND (T) constructs resulted in delayed hatch, and the vasa and nanos constructs had minimal effects on time of hatch (P &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.05). Cadmium chloride appeared to counteract the slow development caused by the TF constructs in two TF treatments…
The masou salmon Δ5-desaturase-like gene (D5D) driven by the common carp β-actin promoter was tra... more The masou salmon Δ5-desaturase-like gene (D5D) driven by the common carp β-actin promoter was transferred into common carp (Cyprinus carpio) that were fed two diets. For P1 transgenic fish fed a commercial diet, Δ6-desaturase-like gene (D6D) and stearoyl-CoA desaturase (SCD) mRNA levels in muscle were up-regulated (P &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.05) 12.7- and 17.9-fold, respectively, and the D6D mRNA level in the gonad of transgenic fish was up-regulated 6.9-fold (P &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.05) compared to that of non-transgenic fish. In contrast, D6D and SCD mRNA levels in transgenic fish were dramatically down-regulated (P &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.05), 50.2- and 16.7-fold in brain, and 5.4- and 2.4-fold in liver, respectively, in comparison with those of non-transgenic fish. When fed a specially formulated diet, D6D and SCD mRNA levels in muscle of transgenic fish were up-regulated (P &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.05) 41.5- and 8.9-fold, respectively, and in liver 6.0- and 3.3-fold, respectively, compared to those of non-transgenic fish. In contrast, D6D and SCD mRNA levels in the gonad of transgenic fish were down-regulated (P &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.05) 5.5- and 12.4-fold, respectively, and D6D and SCD mRNA levels in the brain were down-regulated 14.9- and 1.4-fold (P &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.05), respectively, compared to those of non-transgenic fish. The transgenic common carp fed the commercial diet had 1.07-fold EPA, 1.12-fold DPA, 1.07-fold DHA, and 1.07-fold higher observed total omega-3 fatty acid levels than non-transgenic common carp. Although these differences were not statistically different (P &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;gt; 0.05), there were significantly (P &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.10) higher omega-3 fatty acid levels when considering the differences for all of the individual omega-3 fatty acids. The genotype × diet interactions observed indicated that the potential of desaturase transgenesis cannot be realized without using a well-designed diet with the needed amount of substrates.
<p>Representative channel catfish male-biased genes involved in spermatogenesis, gonad sex ... more <p>Representative channel catfish male-biased genes involved in spermatogenesis, gonad sex determination, and testicular determination.</p
<p>Note that the proportion of male-biased genes per linkage group is similar except for ch... more <p>Note that the proportion of male-biased genes per linkage group is similar except for chromosomes 1, 6, 8, 7, 24 and 27 (shown in red for linkage groups with high numbers of male-biased genes and in yellow for linkage groups with low numbers of male-biased genes).</p
<p>GO annotations were based on zebrafish RefSeq and GO-terms were processed by Blast2Go an... more <p>GO annotations were based on zebrafish RefSeq and GO-terms were processed by Blast2Go and categorized at level 2 into three major functional categories (biological process, cellular component, and molecular function).</p
Stem cells are a class of undifferentiated cells, have the potential for self-renewal that can di... more Stem cells are a class of undifferentiated cells, have the potential for self-renewal that can differ to the specialized cells. First studies on stem cells in fish started with zebra fish in 1992. In this review, classification, resources, vital importance, characteristics and application areas of fish stem cell were clarified.
Abstract Constructs bearing growth hormone (GH) cDNA from channel catfish, Ictalurus punctatus, d... more Abstract Constructs bearing growth hormone (GH) cDNA from channel catfish, Ictalurus punctatus, driven by the ocean pout, Zoarces americanus, antifreeze protein promoter (opAFP), or by the rainbow trout, Oncorhynchus mykiss, metallothionein promoter (rtMT) were transferred to channel catfish via electroporation. Transgenic P1 individuals were mated to produce F1 individuals that exhibited enhanced growth rate. The inheritance of the transgene by the F1 generation was 10% to 36%. The mean body sizes of F1 transgenic fish were larger than their non-transgenic full-siblings (P 0.05) between transgenic and non-transgenic full-siblings except for one opAFP-ccGH family, although the observed means were smaller for four of six transgenic groups as apparently their relative length was increasing more rapidly than relative weight compared to their respective controls. Examining the data from the current experiment and that from an earlier experiment with the same genetic types, but grown to a smaller size, it appears that the two promoters gave similar results in regards to growth enhancement. The largest individuals were always in the transgenic families, and this along with the family effects suggest that gene transfer coupled with combined individual and family selection might result in larger fish than the GH transgenesis alone. The growth enhancement in the current experiment was better than previous experiments with channel catfish that used RSV-LTR-salmonid GH constructs. Assuming government approval and the same growth to food size, GH transgenic channel catfish would increase farm income by $3421/ha compared to use of non-transgenic channel catfish.
Our aim was to transplant blue catfish germ line stem cells into blastulae of triploid channel ca... more Our aim was to transplant blue catfish germ line stem cells into blastulae of triploid channel catfish embryos to produce interspecific xenogenic catfish. The morphological structure of the gonads of blue catfish (Ictalurus furcatus) in ~ 90- to 100-day-old juveniles, two-year-old juveniles, and mature adults was studied histologically. Both oogonia (12-15 μm, diameter with distinct nucleus 7-8 μm diameter) and spermatogonia (12-15 μm, with distinct nucleus 6-7.5 μm diameter) were found in all ages of fish. The percentage of germ line stem cells was higher in younger blue catfish of both sexes. After the testicular tissue was trypsinized, a discontinuous density gradient centrifugation was performed using 70, 45, and 35% Percoll to enrich the percentage of spermatogonial stem cells (SSCs). Four distinct cell bands were generated after the centrifugation. It was estimated that 50% of the total cells in the top band were type A spermatogonia (diameter 12-15 μm) and type B spermatogoni...
Marine biotechnology (New York, N.Y.), Jan 20, 2018
Repressible knockdown approaches were investigated to manipulate for transgenic sterilization in ... more 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 P 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 F fish, mRNA expr...
Repressible knockdown approaches were investigated for transgenic sterilization in channel catfis... more 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 mR...
Marine biotechnology (New York, N.Y.), Jan 4, 2016
Channel catfish (Ictalurus punctatus) is the most important freshwater aquaculture species in the... more Channel catfish (Ictalurus punctatus) is the most important freshwater aquaculture species in the USA. Genetically enhanced fish that are sterile could both profit the catfish industry and reduce potential environmental and ecological risks. As the first step to generate sterile channel catfish, three sets of zinc finger nuclease (ZFN) plasmids targeting the luteinizing hormone (LH) gene were designed and electroporated into one-cell embryos, different concentrations were introduced, and the Cel-I assay was conducted to detect mutations. Channel catfish carrying the mutated LH gene were sterile, as confirmed by DNA sequencing and mating experiments. The overall mutation rate was 19.7 % for 66 channel catfish, and the best treatment was ZFN set 1 at the concentration 25 μg/ml. To our knowledge, this is the first instance of gene editing of fish via plasmid introduction instead of mRNA microinjection. The introduction of the ZFN plasmids may have reduced mosaicism, as mutated individu...
Testicular germ cells of channel catfish, Ictalurus punctatus, and blue catfish, I. furcatus were... more Testicular germ cells of channel catfish, Ictalurus punctatus, and blue catfish, I. furcatus were separated into four layers with Percoll density gradient centrifugation, containing different cell types (40 % in the first layer were spermatogonial stem cells, SSCs). Expression of seventeen genes was analyzed for cells from different layers by real-time quantitative PCR. Pfkfb4, Urod, Plzf, Integrin6, IntegrinV, Thy1 and Cdh1 genes showed the same expression change pattern in both channel and blue catfish as these genes were down-regulated in the spermatocytes and even more so in spermatids. Plzf and Integrin6 had especially high expression in SSCs and can be used as SSCs specific markers. Sox2 gene was up-regulated in spermatocytes and even more highly up-regulated in spermatids, which indicated it could be a spermatid marker. In contrast to channel catfish, Id4, Smad5 and Prdm14 gene expressions were strongly down-regulated in spermatocyte cells, but up-regulated in spermatid cells...
Channel catfish (Ictalurus punctatus) embryos were electroporated with sterilization constructs t... more Channel catfish (Ictalurus punctatus) embryos were electroporated with sterilization constructs targeting primordial germ cell proteins or with buffer. Some embryos then were treated with repressor compounds, cadmium chloride, copper sulfate, sodium chloride or doxycycline, to prevent expression of the transgene constructs. Promoters included channel catfish nanos and vasa, salmon transferrin (TF), modified yeast Saccharomyces cerevisiae copper transport protein (MCTR) and zebrafish racemase (RM). Knock-down systems were the Tet-off (nanos and vasa constructs), MCTR, RM and TF systems. Knock-down genes included shRNAi targeting 5&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39; nanos (N1), 3&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39; nanos (N2) or dead end (DND), or double-stranded nanos RNA (dsRNA) for overexpression of nanos mRNA. These constructs previously were demonstrated to knock down nanos, vasa and dead end, with the repressors having variable success. Exogenous DNA affected percentage hatch (% hatch), as all 14 constructs, except for the TF dsRNA, TF N1 (T), RM DND (C), vasa DND (C), vasa N1 (C) and vasa N2 (C), had lower % hatch than the control electroporated with buffer. The MCTR and RM DND (T) constructs resulted in delayed hatch, and the vasa and nanos constructs had minimal effects on time of hatch (P &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.05). Cadmium chloride appeared to counteract the slow development caused by the TF constructs in two TF treatments…
The masou salmon Δ5-desaturase-like gene (D5D) driven by the common carp β-actin promoter was tra... more The masou salmon Δ5-desaturase-like gene (D5D) driven by the common carp β-actin promoter was transferred into common carp (Cyprinus carpio) that were fed two diets. For P1 transgenic fish fed a commercial diet, Δ6-desaturase-like gene (D6D) and stearoyl-CoA desaturase (SCD) mRNA levels in muscle were up-regulated (P &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.05) 12.7- and 17.9-fold, respectively, and the D6D mRNA level in the gonad of transgenic fish was up-regulated 6.9-fold (P &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.05) compared to that of non-transgenic fish. In contrast, D6D and SCD mRNA levels in transgenic fish were dramatically down-regulated (P &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.05), 50.2- and 16.7-fold in brain, and 5.4- and 2.4-fold in liver, respectively, in comparison with those of non-transgenic fish. When fed a specially formulated diet, D6D and SCD mRNA levels in muscle of transgenic fish were up-regulated (P &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.05) 41.5- and 8.9-fold, respectively, and in liver 6.0- and 3.3-fold, respectively, compared to those of non-transgenic fish. In contrast, D6D and SCD mRNA levels in the gonad of transgenic fish were down-regulated (P &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.05) 5.5- and 12.4-fold, respectively, and D6D and SCD mRNA levels in the brain were down-regulated 14.9- and 1.4-fold (P &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.05), respectively, compared to those of non-transgenic fish. The transgenic common carp fed the commercial diet had 1.07-fold EPA, 1.12-fold DPA, 1.07-fold DHA, and 1.07-fold higher observed total omega-3 fatty acid levels than non-transgenic common carp. Although these differences were not statistically different (P &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;gt; 0.05), there were significantly (P &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.10) higher omega-3 fatty acid levels when considering the differences for all of the individual omega-3 fatty acids. The genotype × diet interactions observed indicated that the potential of desaturase transgenesis cannot be realized without using a well-designed diet with the needed amount of substrates.
Uploads
Papers by dayan perera