Transgenic Progeny Research Articles

Effect of Polyphenol Composition BP-C2 on the Rate of Liver Tumor Reversion of Transgenic Progeny of Irradiated Fish Danio Rerio

Effect of Polyphenol Composition BP-C2 on the Rate of Liver Tumor Reversion of Transgenic Progeny of Irradiated Fish Danio Rerio

Simplified homology-assisted CRISPR for gene editing in Drosophila.

In vivo genome editing with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 generates powerful tools to study gene regulation and function. We revised the homology-assisted CRISPR knock-in method to convert Drosophila GAL4 lines to LexA lines using a new universal knock-in donor strain. A balancer chromosome-linked donor strain with both body color (yellow) and eye red fluorescent protein (RFP) expression markers simplified the identification of LexA knock-in using light or fluorescence microscopy. A second balancer chromosome-linked donor strain readily converted the second chromosome-linked GAL4 lines regardless of target location in the cis-chromosome but showed limited success for the third chromosome-linked GAL4 lines. We observed a consistent and robust expression of the yellow transgene in progeny harboring a LexA knock-in at diverse genomic locations. Unexpectedly, the expression of the 3xP3-RFP transgene in the "dual transgene" cassette was significantly increased compared with that of the original single 3xP3-RFP transgene cassette in all tested genomic locations. Using this improved screening approach, we generated 16 novel LexA lines; tissue expression by the derived LexA and originating GAL4 lines was similar or indistinguishable. In collaboration with 2 secondary school classes, we also established a systematic workflow to generate a collection of LexA lines from frequently used GAL4 lines.

Pyramided transgenic jute (Corchorus capsularis) with biotic stress resistance and herbicide tolerance

Jute (Corchorus sp.) is a commercially important bast (stem) fibre–producing crop. The natural, long, lustrous, and strong jute fibres are globally popular for their domestic and industrial utility. Unfortunately, jute fibre production is under continuous threat from three major agricultural challenges: insect infestation (predominantly lepidopteran insects), fungal infection, and weed management issues. Creating jute plants that could themselves overcome these three challenges would be spectacular. Hence, multi–trait transgenic lines were developed by incorporating three genes—cry1Ab/Ac (encodes Bt δ–endotoxin for Lepidopteran insect resistance), chi11 (encodes chitinase for fungus resistance), and bar (encodes phosphinothricin acetyltransferase (PAT) for glufosinate herbicide tolerance)—into the white jute (C. capsularis) cultivar JRC–321 through Agrobacterium–mediated co–transformation. A bioassay–based event selection strategy was applied for the selection of the transformed jute lines. Each plant was tested through consecutive bioassays involving herbicide, insects, and fungus, followed by molecular and biochemical validation. The functions of the three genes, incorporated in the greenhouse–grown transgenic progeny plants, were confirmed for every generation (till the T2 generation). The plants were able to detoxify the glufosinate herbicide Basta®, resist the two devastating lepidopteran pests tested: hairy caterpillars (Spilarctia obliqua) and semiloopers (Anomis sabulifera), and recover from the stem rot–causing fungal pathogen Macrophomina phaseolina, without compromising agronomic characteristics or fibre quality. The white jute lines thus developed may be effectively used to increase the production of quality jute fibres, meet local and global demands, and minimise input cultivation costs.

An Optimized Small-Scale Rearing System to Support Embryonic Microinjection Protocols for Western Corn Rootworm, Diabrotica virgifera virgifera.

Western corn rootworm (WCR), a major pest of corn, has been reared in laboratories since the 1960s. While established rearing methods are appropriate for maintaining WCR colonies, they are not optimal for performing germline transformation or CRISPR/Cas9-based genome editing. Here we report the development of an optimized rearing system for use in WCR functional genomics research, specifically the development of a system that facilitates the collection of preblastoderm embryos for microinjection as well as gathering large larvae and pupae for downstream phenotypic screening. Further, transgenic-based experiments require stable and well-defined survival rates and the ability to manipulate insects at every life stage. In our system, the WCR life cycle (egg to adult) takes approximately 42 days, with most individuals eclosing between 41 and 45 days post oviposition. Over the course of one year, our overall survival rate was 67%. We used this data to establish a quality control system for more accurately monitoring colony health. Herein, we also offer detailed descriptions for setting up single-pair crosses and conducting phenotypic screens to identify transgenic progeny. This study provides a model for the development of new rearing systems and the establishment of highly controlled processes for specialized purposes.

Development of a Transparent Transgenic Zebrafish Cellular Phenotype Tg(6xNF-kB:EGFP); Casper(roy-/-, nacre-/-) to Study NF-kB Activity.

NF-κB signaling has broad effects on cell survival, tissue growth, and proliferation activities. It controls many genes that are involved in inflammation and thus is a key player in many inflammatory diseases. The elevation of NF-κB activators is associated with elevated mortality, especially in cancer and cardiovascular diseases. The zebrafish has emerged as an important model for whole-organism in vivo modeling in translational research. In vertebrates, in-vivo spatial resolution is limited due to normal opacification of skin and subdermal structure. For in vivo imaging, skin transparency by blocking the pigmentation via chemical inhibition is required and the maintenance of this transparency is vital. The Casper(roy-/-, nacre-/-) mutant of zebrafish maintains this transparency throughout its life and serves as an ideal combination of sensitivity and resolution for in vivo stem cell analyses and imaging. We developed an NF-kB:GFP/Casper transparent transgenic zebrafish cellular phenotype to study inflammatory processes in vivo. We outline the experimental setup to generate a transparent transgenic NF-kB/Casper strain of zebrafish through the cross-breeding of Casper and NF-kB transgenic adult fish and have generated F01 in the form of heterozygous progeny. The transgenic F01 progeny was further inbred to generate heterozygous progenies from F1 to F4 generations. Furthermore, it continued to successfully develop the homozygous strain Tg(6xNF-kB:EGFP); Casper(roy-/-, nacre-/-) in the F05 generation. This novel strain of F05 generation showed 100% homozygosity in the transgenic transparent progeny of Tg(6xNF-kB:EGFP); Casper(roy-/-, nacre-/-). The strain has been confirmed by generating the F06 generation of homozygous progeny and again verified and validated for its homogeneity in the F07 generation. The newly developed novel transparent transgenic strain of the NF-kB reporter line has been coined as "Tg(6xNF-kB:EGFP); Casper(roy-/-, nacre-/-)gmc1". We have established a newly generated phenotype of transparent transgenic zebrafish for time-lapse in vivo confocal microscopy to study the cellular phenotype and pathologies at the cellular level over time. This will allow for quantifying the changes in the NF-kB functional activities over time and allow the comparison of control and cardiac-oncology experimental therapeutics. We validated the newly developed Tg(6xNF-kB:EGFP); Casper(roy-/-, nacre-/-)gmc1 homozygous strain of zebrafish by studying the inflammatory response to bacterial lipopolysaccharide (LPS) exposure, tolerance, and the inhibitory role of a potential novel drug candidate against LPS-induced inflammation. The results establish the unique application of newly developed strains by identifying hit and lead drug candidates for experimental therapeutics.

Serial Recombineering Cloning to Build Selectable and Tagged Genomic P[acman] BAC Clones for Selection Transgenesis and Functional Gene Analysis using Drosophila melanogaster.

Transgenes with genomic DNA fragments that encompass genes of interest are the gold standard for complementing null alleles in rescue experiments in the fruit fly Drosophila melanogaster. Of particular interest are genomic DNA clones available as bacterial artificial chromosomes (BACs) or fosmids from publicly available genomic DNA libraries. Genes contained within BAC and fosmid clones can be easily modified by recombineering cloning to insert peptide or protein tags to localize, visualize, or manipulate gene products, and to create point mutations or deletions for structure-function analysis of the inserted genes. However, since transgenesis efficiency is inversely correlated with transgene size, obtaining transgenic animals for increasingly larger BAC and fosmid clones requires increasingly laborious screening efforts using the transgenesis marker commonly used for these transgenes, the dominant eye color marker white+ . We recently described a drug-based selectable genetic platform for Drosophila melanogaster, which included four resistance markers that allow direct selection of transgenic animals, eliminating the need to identify transgenic progeny by laborious phenotypic screening. By integrating these resistance markers into BAC transgenes, we were able to isolate animals containing large transgenes by direct selection, avoiding laborious screening. Here we present procedures on how to upgrade BAC clones by serial recombineering cloning to build both selectable and tagged BAC transgenes, for selection transgenesis and functional gene analysis, respectively. We illustrate these procedures using a BAC clone encompassing the gene encoding the synaptic vesicle protein, cysteine string protein. We demonstrate that the modified BAC clone, serially recombineered with a selectable marker for selection transgenesis and an N-terminal green fluorescent protein tag for gene expression analysis, is functional by showing the expression pattern obtained after successful selection transgenesis. The protocols cover: (1) cloning and preparation of the recombineering templates needed for serial recombineering cloning to incorporate selectable markers and protein tags; (2) preparing electrocompetent cells needed to perform serial recombineering cloning; and (3) the serial recombineering workflow to generate both selectable and tagged genomic BAC reporter transgenes for selection transgenesis and functional gene analysis in Drosophila melanogaster. The protocols we describe can be easily adapted to incorporate any of four selectable markers, protein tags, or any other modification for structure-function analysis of the genes present within any of the BAC or fosmid clones. A protocol for generating transgenic animals using serially recombineered BAC clones is presented in an accompanying Current Protocols article (Venken, Matinyan, Gonzalez, & Dierick, 2023a). © 2023 Wiley Periodicals LLC. Basic Protocol 1: Cloning and preparation of recombineering templates used for serial recombineering cloning. Basic Protocol 2: Making electrocompetent cells of the bacterial strains used to perform serial recombineering cloning or induction of plasmid copy number. Basic Protocol 3: Serial recombineering cloning to generate both selectable and tagged genomic P[acman] BAC reporter transgenes for selection transgenesis and gene expression analysis in Drosophila melanogaster.

Development of CRISPR/Cas9-Mediated Gene-Drive Construct Targeting the Phenotypic Gene in Plutella xylostella.

The gene-drive system can ensure that desirable traits are transmitted to the progeny more than the normal Mendelian segregation. The clustered regularly interspersed palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) mediated gene-drive system has been demonstrated in dipteran insect species, including Drosophila and Anopheles, not yet in other insect species. Here, we have developed a single CRISPR/Cas9-mediated gene-drive construct for Plutella xylostella, a highly-destructive lepidopteran pest of cruciferous crops. The gene-drive construct was developed containing a Cas9 gene, a marker gene (EGFP) and a gRNA sequence targeting the phenotypic marker gene (Pxyellow) and site-specifically inserted into the P. xylostella genome. This homing-based gene-drive copied ∼12 kb of a fragment containing Cas9 gene, gRNA, and EGFP gene along with their promoters to the target site. Overall, 6.67%–12.59% gene-drive efficiency due to homology-directed repair (HDR), and 80.93%–86.77% resistant-allele formation due to non-homologous-end joining (NHEJ) were observed. Furthermore, the transgenic progeny derived from male parents showed a higher gene-drive efficiency compared with transgenic progeny derived from female parents. This study demonstrates the feasibility of the CRISPR/Cas9-mediated gene-drive construct in P. xylostella that inherits the desired traits to the progeny. The finding of this study provides a foundation to develop an effective CRISPR/Cas9-mediated gene-drive system for pest control.

Natural variants and editing events provide insights into routes for spike architecture modification in common wheat

Spike architecture is an indicative trait of grain yield in common wheat (Triticum aestivum). A segregating population was generated for mapping genes contributing to spike morphometric traits by crossing the two common wheat cultivars 'CItr 17600' with branching spikes and 'Yangmai 18' with normal spikes. A major quantitative trait locus for spike length was mapped to the Q5A region of chromosome 5A. Yangmai 18 carried a Q5Ab allele for short spikes, which harbored one SNP in the last intron, and a 1-bp InDel in the 720-bp fragment from the start codon, compared to Q5Aa in Chinese Spring. CItr 17600 harbored a q5Ab allele for long spikes, which has a 6-bp deletion compared to the reported q5Aa allele that was involved in the binding site of microRNA 172 (miR172). This 6-bp deletion in immediately upstream of this binding site was involved in changes of four amino acids. The natural q5A allele appeared to be rare in common wheat but frequent in tetraploid T. turgidum accessions with branching spikes. The CRISPR/Cas9 technology was used to edit the upstream region involving in the miR172 binding site in Yangmai 18 and identified two independent editing events, one with a 1-bp insertion in Q5A and the other with a 2-bp deletion in Q5D, resulting in several shapes of spikes in the transgenic progeny. In addition to the effects of natural q5A allele and the edited Q5A genes, this study indicated the regeneratability and transformability of Yangmai 18 as an elite cultivar. Altogether, this study provides insight into future modification and engineering of spike architecture in common wheat.

Generating Transgenics and Knockouts in Strongyloides Species by Microinjection.

The genus Strongyloides consists of multiple species of skin-penetrating nematodes with different host ranges, including Strongyloides stercoralis and Strongyloides ratti. S.stercoralis is a human-parasitic, skin-penetrating nematode that infects approximately 610 million people, while the rat parasite S.ratti is closely related to S.stercoralis and is often used as a laboratory model for S.stercoralis. Both S.stercoralis and S.ratti are easily amenable to the generation of transgenics and knockouts through the exogenous nucleic acid delivery technique of intragonadal microinjection, and as such, have emerged as model systems for other parasitic helminths that are not yet amenable to this technique. Parasitic Strongyloides adults inhabit the small intestine of their host and release progeny into the environment via the feces. Once in the environment, the larvae develop into free-living adults, which live in feces and produce progeny that must find and invade a new host. This environmental generation is unique to the Strongyloides species and similar enough in morphology to the model free-living nematode Caenorhabditis elegans that techniques developed for C. elegans can be adapted for use with these parasitic nematodes, including intragonadal microinjection. Using intragonadal microinjection, a wide variety of transgenes can be introduced into Strongyloides. CRISPR/Cas9 components can also be microinjected to create mutant Strongyloides larvae. Here, the technique of intragonadal microinjection into Strongyloides, including the preparation of free-living adults, the injection procedure, and the selection of transgenic progeny, is described. Images of transgenic Strongyloides larvae created using CRISPR/Cas9 mutagenesis are included. The aim of this paper is to enable other researchers to use microinjection to create transgenic and mutant Strongyloides.

Abstract P303: Development Of Casper/myl7/annexin-5 Transparent Transgenic In-vivo Zebrafish Model To Study The Cardiomyocyte Function

The Zebrafish provided an excellent platform to study the genetic and molecular approach ofcardiac research. Zebrafish heart cells similar to human heart cells at the molecular level anddetermine gene functions that control cardiac function and dysfunction. In zebrafish heart, myl7is myosin 7 gene and identified as a regulatory gene orthologs to human MYL7. In the heart,Annexin5 activities contribute to cardiomyocyte dedifferentiation, proliferation and epicardial injuryresponses which leads to cardiac cell death by apoptosis and narcosis pathways. We aredeveloping annexin-5 activity in the cardiovascular function under normal and in metabolicaberration by generating homozygous Casper/ myl7:RFP; annexin-5:YFP transgenic zebrafish.By developing Casper/myl7/Annexin-5 transparent transgenic zebrafish model, we establish time-lapse in-vivo confocal microscopy to study of cellular phenotype/pathologies of thecardiomyocytes over time in newly developed strain to quantify changes in cardiomyocytemorphology and function overtime, comparing control and cardiac injury and cardio-oncologymodels. Transgenic zebrafish has normal type skin pigmentation background. In zebrafish,tracking of transgenic reporter activity in in-vivo is only possible in transparent stage. To maintaintransparency throughout the life, these strains crossbred with the skin transparent mutant Casper.Casper contributes to the study by integrating a transparent characteristic in adult zebrafish thatallows for simpler transparent visualization and observation. We develop casper transgenicprogenies through cross breeding with the transgenic strain of myl7:RFP;annexin-5:YFP .Confocal and fluorescent microscopy used to get accurate, precise imaging and to determinefluorescent protein being activated. 1.1: Generation of homozygous casper / myl7:RFP;annexin-5:YFP zebrafish (Generation F01-F05). 1.2: Screening and sorting the transgenic progeny andIn vivo imaging to validate cardiac morphology through in-vivo confocal imaging. Generation ofhomozygous casper / myl7:RFP;annexin-5:YFP zebrafish: Casper-Annexin5 homozygous stain:Cross breed casper and myl7/Annexin5 fish; F01: Generate the eggs from breeder and grow theembryo to attenuate larvae to screen for transgenic expression. F01 generation, larvae showtransgenic expression (47%). F02: transgenic expression larvae (39%). F02 heterozygous shownormal skin pattern; F03, larval show transgenic expression (43%). F04, transgenic larvae(90%).F04; 100% fishes are phenotypically casper; F05: heterozygous transgenic progeny togrow and continue to generate until achieve 100% homozygous casper-myl7-Annexin5 strain.These novel results provide in-vivo whole organism-based platform to design high throughputscreening and establish new horizon for drug discovery in the Cardiac Disease and Cardio-oncology.

OsATL38 mediates mono-ubiquitination of the 14-3-3 protein OsGF14d and negatively regulates the cold stress response in rice.

One of the major regulatory pathways that permits plants to convert an external stimulus into an internal cellular response within a short period of time is the ubiquitination pathway. In this study, OsATL38 was identified as a low temperature-induced gene that encodes a rice homolog of Arabidopsis Tóxicos en Levadura RING-type E3 ubiquitin (Ub) ligase, which was predominantly localized to the plasma membrane. OsATL38-overexpressing transgenic rice plants exhibited decreased tolerance to cold stress as compared with wild-type rice plants. In contrast, RNAi-mediated OsATL38 knockdown transgenic progeny exhibited markedly increased tolerance to cold stress relative to that of wild-type plants, which indicated a negative role of OsATL38 in response to cold stress. Yeast two-hybrid, in vitro pull-down, and co-immunoprecipitation assays revealed that OsATL38 physically interacted with OsGF14d, a rice 14-3-3 protein. An in vivo target ubiquitination assay indicated that OsGF14d was mono-ubiquitinated by OsATL38. osgf14d knockout mutant plants were more sensitive to cold stress than wild-type rice plants, indicating that OsGF14d is a positive factor in the response to cold stress. These results provide evidence that the RING E3 Ub ligase OsATL38 negatively regulates the cold stress response in rice via mono-ubiquitination of OsGF14d 14-3-3 protein.

Development of Tg(UAS:SEC-Hsa.ANXA5-YFP,myl7:RFP); Casper(roy-/-,nacre-/-) Transparent Transgenic In Vivo Zebrafish Model to Study the Cardiomyocyte Function.

The zebrafish provided an excellent platform to study the genetic and molecular approach of cellular phenotype-based cardiac research. We designed a novel protocol to develop the transparent transgenic zebrafish model to study annexin-5 activity in the cardiovascular function by generating homozygous transparent skin Casper(roy−/−,nacre−/−); myl7:RFP; annexin-5:YFP transgenic zebrafish. The skin pigmentation background of any vertebrate model organism is a major obstruction for in vivo confocal imaging to study the transgenic cellular phenotype-based study. By developing Casper(roy−/−,nacre−/−); myl7; annexin-5 transparent transgenic zebrafish strain, we established time-lapse in vivo confocal microscopy to study cellular phenotype/pathologies of cardiomyocytes over time to quantify changes in cardiomyocyte morphology and function over time, comparing control and cardiac injury and cardio-oncology. Casper contributes to the study by integrating a transparent characteristic in adult zebrafish that allows for simpler transparent visualization and observation. The Casper(roy−/−,nacre−/−) transgenic progenies developed through cross-breeding with the transgenic strain of Tg(UAS:SEC-Hsa.ANXA5-YFP,myl7:RFP). Confocal and fluorescent microscopy were being used to obtain accurate, precise imaging and to determine fluorescent protein being activated. This study protocol was conducted under two sections; 1.1: Generation of homozygous Tg(UAS:SEC-Hsa.ANXA5-YFP,myl7:RFP); Casper(roy−/−,nacre−/−) zebrafish (generation F01-F06) and 1.2: Screening and sorting the transparent transgenic progeny and in vivo imaging to validate cardiac morphology through in vivo confocal imaging. We coined the newly developed strain as Tg(UAS:SEC-Hsa.ANXA5-YFP,myl7:RFP); Casper(roy−/−,nacre−/−)gmc1. Thus, the newly developed strain maintains transparency of the skin throughout the entire life of zebrafish and is capable of application of a non-invasive in vivo imaging process. These novel results provide an in vivo whole organism-based platform to design high-throughput screening and establish a new horizon for drug discovery in cardiac cell death and cardio-oncology therapeutics and treatment.

Generation of Marker-Free Transgenic Rice Resistant to Rice Blast Disease Using Ac/Ds Transposon-Mediated Transgene Reintegration System.

Rice blast is one of the most serious diseases of rice and a major threat to rice production. Breeding disease-resistant rice is one of the most economical, safe, and effective measures for the control of rice blast. As a complement to traditional crop breeding, the transgenic method can avoid the time-consuming process of crosses and multi-generation selection. In this study, maize (Zea mays) Activator (Ac)/Dissociation (Ds) transposon vectors carrying green fluorescent protein (GFP) and red fluorescent protein (mCherry) genetic markers were used for generating marker-free transgenic rice. Double fluorescent protein-aided counterselection against the presence of T-DNA was performed together with polymerase chain reaction (PCR)-based positive selection for the gene of interest (GOI) to screen marker-free progeny. We cloned an RNAi expression cassette of the rice Pi21 gene that negatively regulates resistance to rice blast as a GOI into the Ds element in the Ac/Ds vector and obtained marker-free T1 rice plants from 13 independent transgenic lines. Marker-free and Ds/GOI-homozygous rice lines were verified by PCR and Southern hybridization analysis to be completely free of transgenic markers and T-DNA sequences. qRT-PCR analysis and rice blast disease inoculation confirmed that the marker-free transgenic rice lines exhibited decreased Pi21 expression levels and increased resistance to rice blast. TAIL-PCR results showed that the Ds (Pi21-RNAi) transgenes in two rice lines were reintegrated in intergenic regions in the rice genome. The Ac/Ds vector with dual fluorescent protein markers offers more reliable screening of marker-free transgenic progeny and can be utilized in the transgenic breeding of rice disease resistance and other agronomic traits.

DNA Methylation Silences Exogenous Gene Expression in Transgenic Birch Progeny.

The genetic stability of exogenous genes in the progeny of transgenic trees is extremely important in forest breeding; however, it remains largely unclear. We selected transgenic birch (Betula platyphylla) and its hybrid F1 progeny to investigate the expression stability and silencing mechanism of exogenous genes. We found that the exogenous genes of transgenic birch could be transmitted to their offspring through sexual reproduction. The exogenous genes were segregated during genetic transmission. The hybrid progeny of transgenic birch WT1×TP22 (184) and WT1×TP23 (212) showed higher Bgt expression and greater insect resistance than their parents. However, the hybrid progeny of transgenic birch TP23×TP49 (196) showed much lower Bgt expression, which was only 13.5% of the expression in its parents. To elucidate the mechanism underlying the variation in gene expression between the parents and progeny, we analyzed the methylation rates of Bgt in its promoter and coding regions. The hybrid progeny with normally expressed exogenous genes showed much lower methylation rates (0–29%) than the hybrid progeny with silenced exogenous genes (32.35–45.95%). These results suggest that transgene silencing in the progeny is mainly due to DNA methylation at cytosine residues. We further demonstrated that methylation in the promoter region, rather than in the coding region, leads to gene silencing. We also investigated the relative expression levels of three methyltransferase genes: BpCMT, BpDRM, and BpMET. The transgenic birch line 196 with a silenced Gus gene showed, respectively, 2.54, 9.92, and 4.54 times higher expression levels of BpCMT, BpDRM, and BpMET than its parents. These trends are consistent with and corroborate the high methylation levels of exogenous genes in the transgenic birch line 196. Therefore, our study suggests that DNA methylation in the promoter region leads to silencing of exogenous genes in transgenic progeny of birch.

Homozygous Transgenic Barley (Hordeum vulgare L.) Plants by Anther Culture.

Production of homozygous lines derived from transgenic plants is one of the important steps for phenotyping and genotyping transgenic progeny. The selection of homozygous plants is a tedious process that can be significantly shortened by androgenesis, cultivation of anthers, or isolated microspores. Doubled haploid (DH) production achieves complete homozygosity in one generation. We obtained transgenic homozygous DH lines from six different transgenic events by using anther culture. Anthers were isolated from T0 transgenic primary regenerants and cultivated in vitro. The ploidy level was determined in green regenerants. At least half of the 2n green plants were transgenic, and their progeny were shown to carry the transgene. The process of dihaploidization did not affect the expression of the transgene. Embryo cultures were used to reduce the time to seed of the next generation. The application of these methods enables rapid evaluation of transgenic lines for gene function studies and trait evaluation.

Establishment of the 'imbibed seed piercing' method for Agrobacterium-mediated transformation of jute and flax bast fibre crops via phloem-specific expression of the β-glucuronidase Gene

Jute (Corchorus capsularis) and flax (Linum usitatissimum) have been established as important sources of bast fibres (phloem fibres) worldwide. These long and lustrous natural fibres are used for various commercial and industrial purposes and hold immense future prospects. To meet the worldwide demand for bast fibres, improvement in the production and quality of jute and flax is essential. Molecular breeding is an expeditious approach for varietal improvement and requires an efficient gene transformation system. The available methods for jute and flax transformation are highly specific to species and variety, requiring a long time and rigorous preparation of media, explants, and screening procedures. In this study, we used a fast and stable method for Agrobacterium tumefaciens-mediated transformation of jute (variety JRC321) and flax (variety FT-897), known as the ‘imbibed seed piercing’ method (ISPM), where seeds were used as explants. A phloem-specific Arabidopsis sucrose-proton H+ symporter 2 (AtSUC2) promoter-driven gus gene (β-glucuronidase) was used for the assessment of genetic transformation of jute and flax plants. Transformed seedlings were selected following three consecutive selection pressure tests conducted at two-week intervals using kanamycin-supplemented culture media. Transgene integration was verified using Southern blotting and PCR-based molecular characterization of jute and flax plants. The gus gene expression levels in the transgenic jute and flax tissue samples were analysed by semi-quantitative and quantitative RT-PCR. The GUS protein expression in transgenic plant tissues was confirmed by the development of blue colour in the histochemical GUS assay. Blue colouration was observed only in phloem cells of the tested plant tissues. Transgenic progeny plants of subsequent generations followed the 3:1 transgene segregation pattern and exhibited phloem-specific GUS expression. This fast, easy, and stable ISPM could also be applied to other fibre producing industrial crops and could form a part of a modern CRISPR/Cas9-based genome editing tool by efficiently delivering CRISPR vectors in jute and flax plants.

In vivoimaging of transgenic Brugia malayi.

BackgroundStudies of the human filarial parasite have been hampered by the fact that they are obligate parasites with long life cycles. In other pathogenic infections, in vivo imaging systems (IVIS) have proven extremely useful in studying pathogenesis, tissue tropism and in vivo drug efficacy. IVIS requires the use of transgenic parasites expressing a florescent reporter. Developing a method to produce transgenic filarial parasites expressing a florescent reporter would permit IVIS to be applied to the study of tissue tropism and provide a non-invasive way to screen for in vivo drug efficacy against these parasites.Methodology/Principal findingsWe report the development of a dual luciferase reporter construct in a piggyBac backbone that may be used to stably transfect Brugia malayi, a causative agent of human filariasis. Parasites transfected with this construct were visible in IVIS images obtained from infected gerbils. The signal in these infected animals increased dramatically when the transgenic parasites matured to the adult stage and began to produce transgenic progeny microfilaria. We demonstrate that the IVIS system can be used to develop an effective method for cryopreservation of transgenic parasites, to non-invasively monitor the effect of treatment with anti-filarial drugs, and to rapidly identify transgenic F1 microfilariae.ConclusionsTo our knowledge, this represents the first application of IVIS to the study of a human filarial parasite. This method should prove useful in studies of tissue tropism and as an efficient in vivo assay for candidate anti-filarial drugs.

The efficiency of use lentiviral vectors for the transformation of rooster spermatogenic cells in vivo

Male gonad cells are considered as promising target cells for the introduction of recombinant DNA within obtaining genetically modified individuals with given characteristics. The use of testicular spermatogonial stem cells is of the greatest interest. In the process of differentiation, this type of cell gives rise to a significant population of mature male germ cells. In the case of their genetic transformation, differentiated cells can be used to inseminate females in order to produce transgenic progeny. The aim of the research was to study the efficiency of using lentiviral vectors for the local transformation of roosters’ testicular spermatogenic cells. We used a lentiviral vector containing the ZsGreen reporter gene under the control of the CMV promoter. In vitro transformation of rooster spermatogenic cells was carried out by infection with a viral preparation, in vivo through multiple injections of the viral preparation into the testicular parenchyma of roosters ( n = 5). The efficiency of transformation was assessed by expression of the reporter ZsGreen gene in transfected spermatogenic cells. The success of using lentiviral vectors for the genetic transformation of rooster spermatogenic cells was shown in experiments in vitro and in vivo . The transformation efficiency of this cells types in an in vitro culture varied from 45 to 57% and averaged 48 ± 4%. The expression of the ZsGreen reporter gene in the cells of the spermatogenic epithelium of the testes was established in almost all experimental roosters in the in vivo experiments. The number of seminiferous tubules with transformed spermatogenic cells varied in the studied experimental roosters from 10 to 22%. The effectiveness of genetic transformation of the testes spermatogenic cells was 1.8 ± 0.2%. The obtained results indicate to the success of using lentiviral vectors for the genetic transformation of spermatogenic cells of rooster testes in vivo in order to create individuals with genetically transformed germ cells for the further production of transgenic offspring with given characteristics.

Genetic Relationship of the Transgenic and Non-Transgenic Mutiara Catfish with their Hybrids Sangkuriang Catfish

The male broodstock of Transgenic Mutiara catfish (P) (containing Clarias gariepinus Growth Hormone, CgGH) is a fish who show fast growth. Propagation of the Transgenic Mutiara catfish F1 and F2 progeny involves the crossing of broodstock P with Non Transgenic Mutiara catfish. New genetic variations (polymorphisms) can be generated from crosses broodstock P and F1 which lead to the fast growth inheritance of transgenic fish. Phylogenetic relationship analysis showed distance and genetic variation in broodstock P, F1, F2 and Sangkuriang catfish (as controls) using the RAPD-PCR method (Random Amplified Polymorphic DNA-Polymerase Chain Reaction) as the research purposes. The results showed that OPA-03 (5'-AGTCAGCCAC-3 ') primer was selective for determining phylogenetic among catfish samples. The closest genetic similarity index originates from the female broodstock of Non Transgenic Mutiara catfish (P) (93%), while the farthest originates from the male broodstock of Mutiara Transgenic catfish (P) (51%) with F1 dan F2. Closely related broodstock catfish is not recommended to be crossed.

Fluorescent protein transgenesis has varied effects on behaviour and cold tolerance in a tropical fish (Gymnocorymbus ternetzi): implications for risk assessment.

Fluorescent protein (FP) transgenesis is used in the ornamental aquarium trade to produce new colour morphs in tropical fish. Understanding whether such genetic modification could alter ability to survive temperate waters, or interactions with native fish, should such fish be released to natural systems is critical in developing policy on their commercial use. We examined the competitive foraging ability and cold tolerance of unrelated pet-trade sourced adult green FP transgenic tetra and non-transgenic white tetra (Gymnocorymbus ternetzi), as well as white non-transgenic and green FP transgenic juvenile progeny of these groups. FP transgenesis did not affect the foraging success or aggressive behaviour in either adult or juvenile fish, indicating FP transgenesis may not influence potential hazards through this pathway. During a cold temperature tolerance trial, adult green tetras had greatly diminished cold tolerance relative to unrelated adult white fish, while sibling juvenile offspring of these groups had intermediate cold tolerance between adult fish groups that were not affected by FP transgenesis. This data suggests background genetics, rearing history and/or life stage may play larger roles in cold tolerance than FP transgenesis in this species. Unexpectedly, both adult and juvenile white tetras were 3.8 times more likely to take refuge in shelters when temperature declined than green tetras. These data indicate FP transgenic fish may pose equal or lesser risk than non-transgenic fish, should they be released to natural environments. Results also demonstrate that unrelated pet-trade sourced fish may not always be appropriate models for examining effects of FP transgenesis.