Preliminary Study on Transgenesis by Injecting Exogenous DNA into Zygote Cytoplasm of Buffalo

[Objective] This study aimed to investigate the feasibility of transgenesis by injecting exogenous DNA into zygote cytoplasm of Buffalo. [Method] Buffalo oocytes were randomly divided into two groups 20-22 h after in vitro maturation. One group of oocytes was introduced with about 7.5 pl of 50 μg/ml DNA solution containing linear EGFP fragment by cytoplasmic injection 7-10 h or 18-20 h after in vitro fertilization （IVF）; the other group of oocytes was introduced with mixture of a single buffalo sperm and about 7.5 pl of 50 μg/ml DNA solution containing linear EGFP fragment by cytoplasmic injection （generally called ICSI-Mediated Gene Transfer, ICSI-Tr）. Expression of exogenous DNA was observed and recorded during the process of embryonic development. [Result] Early embryonic gene expression efficiency and blastocyst gene expression efficiency in IVF injection group showed no significant difference compared with that in ICSI-Tr group （P0.05）. In addition, the cleavage rate and early embryonic gene expression efficiency in IVF injection group were significantly higher with injection at 7-10 h post IVF than that at 18-20 h post IVF （P0.05）. [Conclusion] These results indicate that transgenic buffalo embryos can be generated by injecting exogenous DNA into cytoplasm of IVF oocytes, and the optimal injection time is 7-10 h post IVF.

Primordial germ cell-mediated transgenesis and genome editing in birds

Transgenesis and genome editing in birds are based on a unique germline transmission system using primordial germ cells(PGCs), which is quite different from the mammalian transgenic and genome editing system. PGCs are progenitor cells of gametes that can deliver genetic information to the next generation. Since avian PGCs were first discovered in nineteenth century, there have been numerous efforts to reveal their origin, specification, and unique migration pattern, and to improve germline transmission efficiency. Recent advances in the isolation and in vitro culture of avian PGCs with genetic manipulation and genome editing tools enable the development of valuable avian models that were unavailable before. However, many challenges remain in the production of transgenic and genome-edited birds,including the precise control of germline transmission, introduction of exogenous genes, and genome editing in PGCs.Therefore, establishing reliable germline-competent PGCs and applying precise genome editing systems are critical current issues in the production of avian models. Here, we introduce a historical overview of avian PGCs and their application, including improved techniques and methodologies in the production of transgenic and genome-edited birds, and we discuss the future potential applications of transgenic and genome-edited birds to provide opportunities and benefits for humans.

Development of genome engineering technologies in cattle: from random to specific

The production of transgenic farm animals(e.g., cattle) via genome engineering for the gain or loss of gene functions is an important undertaking. In the initial stages of genome engineering, DNA micro-injection into one-cell stage embryos(zygotes) followed by embryo transfer into a recipient was performed because of the ease of the procedure.However, as this approach resulted in severe mosaicism and has a low efficiency, it is not typically employed in the cattle as priority, unlike in mice. To overcome the above issue with micro-injection in cattle, somatic cell nuclear transfer(SCNT) was introduced and successfully used to produce cloned livestock. The application of SCNT for the production of transgenic livestock represents a significant advancement, but its development speed is relatively slow because of abnormal reprogramming and low gene targeting efficiency. Recent genome editing technologies(e.g.,ZFN, TALEN, and CRISPR-Cas9) have been rapidly adapted for applications in cattle and great results have been achieved in several fields such as disease models and bioreactors. In the future, genome engineering technologies wil accelerate our understanding of genetic traits in bovine and wil be readily adapted for bio-medical applications in cattle.

Manipulation of spermatogonial stem cells in livestock species

We are entering an exciting epoch in livestock biotechnology during which the fundamental approaches(such as transgenesis, spermatozoa cryopreservation and artificial insemination) will be enhanced based on the modern understanding of the biology of spermatogonial stem cells(SSCs) combined with the outstanding recent advances in genomic editing technologies and in vitro cell culture systems. The general aim of this review is to outline comprehensively the promising applications of SSC manipulation that could in the nearest future find practical application in livestock breeding. Here, we will focus on 1) the basics of mammalian SSC biology;2) the approaches for SSC isolation and purification;3) the available in vitro systems for the stable expansion of isolated SSCs;4) a discussion of how the manipulation of SSCs can accelerate livestock transgenesis;5) a thorough overview of the techniques of SSC transplantation in livestock species(including the preparation of recipients for SSC transplantation,the ultrasonographic-guided SSC transplantation technique in large farm animals, and the perspectives to improve further the SSC transplantation efficiency), and finally, 6) why SSC transplantation is valuable to extend the techniques of spermatozoa cryopreservation and/or artificial insemination. For situations where no reliable data have yet been obtained for a particular livestock species, we will rely on the data obtained from studies conducted in rodents because the knowledge gained from rodent research is translatable to livestock species to a great extent. On the other hand, we will draw special attention to situations where such translation is not possible.

Transient expression of the enhanced green fluorescent protein(egfp) gene in Sargassum horneri

Sargassum horneri is a macroalga widespread in North Asia-Pacific region, and these years its bloom has caused huge damage to the environment and the economic in China. To make up the blank on genetic engineering research, a transient transformation system for the multicellular marine brown alga S . horneri was established in this research. The algae used in this research were collected from the Yellow Sea of China and verified as a same species S . horneri with analysis of molecular markers. The S . horneri parietal leaves were transformed with the enhanced green fluorescent gene as the reporter by micro-particle bombardment. The results show that green fluorescent protein (GFP) is an eff ective transgene reporter for S . horneri and that particle bombardment is a suitable method for transformation of S . horneri . Through selection of four diff erent promoters for EGFP and six groups’ bombardment characters, the highest transformation efficiency approximately 1.31% was got with the vector pEGFP-N1 at bombardment characters 900 spi and 6 cm distance. This research paves a way for the further research and application of S . horneri .

Overexpression of the tissue inhibitor of metalloproteinase-3 during Xenopus embryogenesis affects head and axial tissue formation

Tissue inhibitors of metalloproteinases (TIMPs) modulate extracellular matrix remodeling during embryonic develop- ment and disease. TIMP-3 expression was examined during Xenopus laevis embryogenesis: TIMP-3 transcripts detected in the maternal pool of RNA increased at the mid-blastula transition, decreased dramatically during gastrulation and increased again during neurulation and axis elongation. Interestingly, the decrease during gastrulation was not seen in LiCl treated (dorsalized) embryos. Whole mount in situ hybridization of TIMP-3 using DIG-labeled RNA probes demonstrated that the transcripts were present in all dorsal tissues during embryogenesis, but were prominent only in head structures starting at stage 35. Overexpression of TIMP-3 through transgenesis and RNA injections led to devel- opmental abnormalities and death. Both overexpression strategies resulted in post-gastrulation perturbation including those to neural and head structures, as well as truncated axes. However, RNA injections resulted in more severe early defects such as failure of neural tube closure, and transgenesis caused truncated axes and head abnormalities. No transgenic embryo expressing TIMP-3 survived past stage 40.

An efficient strategy for generation of transgenic mice by lentiviral transduction of male germline stem cells in vivo

Background： Male germline stem cells（MGSCs） are a subpopulation of germ cells in the testis tissue. MGSCs are capable of differentiation into spermatozoa and thus are perfect targets for genomic manipulation to generate transgenic animals.Method： The present study was to optimize a protocol of production of transgenic mice through transduction of MGSCs in vivo using lentiviral-based vectors. The recombinant lentiviral vectors with either EF-1 or CMV promoter to drive the expression of enhanced green fluorescent protein（e GFP） transgene were injected into seminiferous tubules or inter-tubular space of 7-day-old and 28-day-old mouse testes. At 5 or 6 wk post-surgery, these pre-founders were mated with wild-type C57BL/6J female mice（1.5 to 2.0-month-old）.Results： Sixty-seven percent of F1 generation and 55.56 % of F2 offspring were positive for eG FP transgene under the control of EF-1 promoter via PCR analysis. The transgenic pups were generated in an injection site-and age-independent manner. The expression of transgene was displayed in the progeny derived from lentiviral vector containing CMV promoter to drive transgene, but it was silenced or undetectable in the offspring derived from lentiviral vector with transgene under EF-1 promoter. The methylation level of g DNA in the promoter region of transgene was much higher in the samples derived lentiviral vectors with EF-1 promoter than that with CMV promoter,suggesting e GFP transgene was suppressed by DNA methylation in vivo.Conclusion： This research reported here an effective strategy for generation of transgenic mice through transduction of MGSCs in vivo using lentivirus vectors with specific promoters, and the transgenic offspring were obtained in an injection site-and age-independent manner. This protocol could be applied to other animal species, leading to advancement of animal transgenesis in agricultural and biomedical fields.

Morphological changes of gonadotropin-releasing hormone neurons in the rat preoptic area across puberty

Gonadotropin-releasing hormone （GnRH） neurons in the preoptic area may undergo morphological changes during the pubertal period when their activities are upregulated. To clarify the regulatory mechanism of puberty onset, this study aimed to investigate the morphological changes of GnRH neurons in the preoptic area of GnRH-enhanced green fluorescent protein transgenic rats. Under confocal laser microscopy, pubertal GnRH neurons exhibited an inverted Y distribution pattern. Prepubertal GnRH neurons were generally unipolar and bipolar, and were distinguished as smooth type cells with few small processes or irregular type cells with many spine-like processes in the proximal dendrites. The number of GnRH neurons in the preoptic area and spine-like processes were increased during the course of reproductive maturation. There was no significant difference between male and female rats. Immunofluorescence staining revealed synaptophysin punctae close to the distal end of GnRH neurons, indicating that some presynaptic terminals may form a synaptic linkage with these neurons.

An Efficient Intragenic Vector for Generating Intragenic and Cisgenic Plants in Citrus

Genetic transformation has become a promising tool for improvement of a variety of crop species. However, transferring genes across species, the presence of selectable marker genes, and bacteria-derived vector backbone sequences have raised considerable health and environmental concerns. Intragenic vector system-based intragenesis/cisgenesis is a new method using transgenic approach to achieving traditional breeding objectives but circumventing many of the associated shortcomings. We report here the development of an intragenic vector by assembling a T-DNA-like fragment and a buffering sequence following the left border from Citrus clementina into the backbone of the binary vector pCB302. Recovery of citrus regenerants is performed under non-selective conditions and positive intra-/cisgenic regenerants were identified through PCR analysis. Transformation efficiencies obtained in Arabidopsis and “Duncan” grapefruit were ~3% and ~0.67%, respectively, demonstrating the potential of the system for development of “foreign DNA-free” intra-/cisgenic citrus cultivars.

The Stripe Rust Resistance Gene Yr10 Encodes an Evolutionary-Conserved and Unique CC-NBS-LRR Sequence in Wheat

The first seedling or all-stage resistance （R） R gene against stripe rust isolated from Moro wheat （Triticum aes- tivum L.） using a map-based cloning approach was identified as Yr10. Clone 4B of this gene encodes a highly evolutionary- conserved and unique CC-NBS-LRR sequence. Clone 4E, a homolog of Yr10, but lacking transcription start site （TSS） and putative TATA-box and CAAT-box, is likely a non-expressed pseudogene. Clones 4B and 4E are 84% identical and divergent in the intron and the LRR domain. Gene silencing and transgenesis were used in conjunction with inoculation with differen- tially avirulent and virulent stripe rust strains to demonstrate Yr10 functionality. The Yr10 CC-NBS-LRR sequence is unique among known CC-NBS-LRR R genes in wheat but highly conserved homologs （E = 0.0） were identified in Aegilops tauschii and other monocots including Hordeum vulgare and Brachypodium distachyon. Related sequences were also identified in genomic databases of maize, rice, and in sorghum. This is the first report of a CC-NBS-LRR resistance gene in plants with limited homologies in its native host, but with numerous homologous R genes in related monocots that are either host or non-hosts for stripe rust. These results represent a unique example of gene evolution and dispersion across species.

Genome editing in Drosophila melanogaster： from basic genome engineering to the multipurpose CRISPR-Cas9 system

Nowadays, genome editing tools are indispensable for studying gene function in order to increase our knowledge of biochemical processes and disease mechanisms. The extensive availability of mutagenesis and transgenesis tools make Drosophila melanogaster an excellent model organism for geneticists. Early mutagenesis tools relied on chemical or physical methods,ethyl methane sulfonate(EMS) and X-rays respectively, to randomly alter DNA at a nucleotide or chromosomal level. Since the discovery of transposable elements and the availability of the complete fly genome, specific genome editing tools, such as P-elements, zinc-finger nucleases(ZFNs) and transcription activator-like effector nucleases(TALENs), have undergone rapid development. Currently, one of the leading and most effective contemporary tools is the CRISPR-cas9 system made popular because of its low cost, effectiveness, specificity and simplicity of use. This review briefly addresses the most commonly used mutagenesis and transgenesis tools in Drosophila, followed by an in-depth review of the multipurpose CRISPR-Cas9 system and its current applications.

Transgenic Nonhuman Primate Models for Human Diseases:Approaches and Contributing Factors

Nonhuman primates （NHPs） provide powerful experimental models to study human development, cognitive functions and disturbances as well as complex behavior, because of their genetic and physiological similarities to humans. Therefore, NHPs are appropriate models for the study of human diseases, such as neurodegenerative diseases including Parkinson＇s, Alzheimer＇s and Huntington＇s diseases, which occur as a result of genetic mutations. However, such diseases afflicting humans do not occur naturally in NHPs. So transgenic NHPs need to be established to understand the etiology of disease pathology and pathogenesis. Compared to rodent genetic models, the generation of transgenic NHPs for human diseases is inefficient, and only a transgenic monkey model for Huntington＇s disease has been reported. This review focuses on potential approaches and contributing factors for generating transgenic NHPs to study human diseases.

The genetic program Of oocytes can be modified in vivo in the zebrafish ovary

Oocytes,the irreplaceable gametes for generating a new organism,are matured in the ovary of living female animals.It is unknown whether any genetic manipulations can be applied to immature oocytes inside the living ovaries.As a proof-of-concept, we here demonstrate genetic amendments of zebrafish immature oocytes within the ovary.Oocyte microinjection in situ (OMIS) stimulates tissue repair responses,but some of the microinjected immature oocytes are matured,ovulated and fertilizable.By OMIS-mediated Cas9 approach,ntla and gata5 loci of oocytes arrested at prophase I of meiosis are successfully edited before fertilization.Through OMIS,high efficiency of biaUeUc mutations in single or multiple loci using Cas9/gRNAs allows immediate manifestation of mutant phenotypes in Fo embryos and multiple transgenes can co-express the reporters in Fo embryos with patterns similar to germline transgenic embryos.Furthermore,maternal knockdown of dnmt1 by antisense morphoino via OMIS results in a dramatic decrease of global DNA methylation level at the dome stage and causes embryonic lethalityprior to segmentation period.Therefore,OMIS opens a door to efficiently modify the genome and provides a possibility to re repair genetically abnormal oocytes in situ.

Biotechnological interventions for the sustainable management of a global pest,whitefly(Bemisia tabaci)

Whiteflies(Bemisia tabaci)are polyphagous invasive hemipteran insects that cause serious losses of important crops by directly feeding on phloem sap and transmitting pathogenic viruses.These insects have emerged as a major threat to global agriculture and food security.Chemically synthesized insecticides are currently the only option to control whiteflies,but the ability of whiteflies to evolve resistance against insecticides has made the management of these insects very difficult.Natural host-plant resistance against whiteflies identified in some crop plants has not been exploited to a great extent.Genetic engineering approaches,such as transgenics and RNA interference(RNAi),are potentially useful for the control of whiteflies.Transgenic plants harboring insecticidal toxins/lectins developed via nuclear or chloroplast transformation are a promising vehicle for whitefly control.Double-stranded RNAs(dsRNAs)of several insect genes,delivered either through microinjection into the insect body cavity or orally via an artificial diet and transiently or stably expressed in transgenic plants,have controlled whiteflies in model plants and in some crops at the laboratory level,but not at the field level.In this review,we highlight the merits and demerits of each delivery method along with strategies for sustained delivery of dsRNAs via fungal entomopathogen/endosymbiont or nontransgenic RNAi approaches,foliar sprays,root absorption or nanocarriers as well as the factors affecting efficient RNAi and their biosafety issues.Genome sequencing and transcriptome studies of whitefly species are facilitating the selection of appropriate genes for RNAi and gene-editing technology for the efficient and resilient management of whiteflies and their transmitted viruses.

Genome Editing:From Drosophila to Non-Model Insects and Beyond

Insect is the largest group of animals on land.Many insect species inflict economical and health losses to humans.Yet many more benefit us by helping to maintain balances in our ecosystem.The benefits that insects offer remain largely untapped,justifying our continuing efforts to develop tools to better understand their biology and to better manage their activities.Here we focus on reviewing the progresses made in the development of genome engineering tools for model insects.Instead of detailed descriptions of the molecular mechanisms underlying each technical advance,we focus our discussion on the logistics for implementing similar tools in non-model insects.Since none of the tools were developed specific for insects,similar approaches can be applied to other non-model organisms.

Functional genomics studies on the innate immunity of disease vectors

The increasing availability of genome sequences and the development of highthroughput techniques for gene expression profiling and functional characterization are transforming the study of innate immunity and other areas of insect biology. Already, functional genomic approaches have enabled a quantum advance in the characterization of mosquito immune responses to malaria parasite infection, and similar high-throughput functional genomic studies of other vector-pathogen interactions can be expected in the near future. The application of microarray-based and other expression analyses provide genomewide transcriptional profiles that can be used to identify insect immune system components that are differentially regulated upon exposure to various classes of pathogens, including many important etiologic agents of human and animal diseases. The role of infection-responsive or other candidate immune genes identified through comparative genomic approaches can then be functionally characterized, either in vivo, for instance in adult mosquitoes, or in vitro using cell lines. In most insect vectors of human pathogens, germ-line transgenesis is still technically difficult and maintenance of multiple transgenic lines logistically demanding. Consequently, transient RNA interference （RNAi）-mediated gene-silencing has rapidly become the method of choice for functional characterization of candidate innate immune genes. The powerful combination of transcriptional profiling in conjunction with assays using RNAi to determine gene function, and identify regulatory pathways, together with downstream cell biological approaches to determine protein localization and interactions, will continue to provide novel insights into the role of insect innate immunity in a variety of vector-pathogen interactions. Here we review advances in functional genomics studies of innate immunity in the insect disease vectors, over the past decade, with a particular focus on the Anopheles mosquito and its responses to malaria infection.

An Open-Source System for In Planta Gene Stacking by Bxbl and Cre Recombinases

The rapid development of crops with multiple transgenic traits arouses the need for an efficient system for creating stacked cultivars. Most major crops rely on classical breeding to introgress the transgene from a laboratory variety to the numerous cultivars adapted to different growing regions. Even with vegetative propagated crops, genetic crosses are conducted during varietal improvement prior to vegetative cloning. The probability to assort the ＇x＇ number of transgenic loci into a single genome may seem trivial, （~）x for a diploid species, but given the ＇y＇ number of other nontransgenic traits that breeders also need to assemble into the same genome, the （~）~＊y probability for a ＇breeding stack＇ could quickly make the line conversion process unmanageable. Adding new transgenes onto existing transgenic varieties without creating a new segregating locus would require site-specific integration of new DNA at the existing transgenic locus. Here, we tested a recombinase-mediated gene-stacking scheme in tobacco. Sequential site-specific inte- gration was mediated by the mycobacteriophage Bxbl integrase-catalyzed recombination between attP and attB sites. Transgenic DNA no longer needed after integration was excised by Cre recombinase-mediated recombination of Iox sites. Site-specific integration occurred in -10% of the integration events, with half of those events usable as substrates for a next round of gene stacking. Among the site-specific integrants, however, a third experienced gene silencing. Overall, precise structure and reproducible expression of the sequentially added triple traits were obtained at an overall rate of -3% of the transformed clones--a workable frequency for the development of commercial cultivars. Moreover, since nei- ther the Bxbl-att nor the Cre-lox system is under patent, there is freedom to operate,

Using chimeric piggyBac transposase to achieve directed interplasmid transposition in silkworm Bombyx mori and fruit fly Drosophila cells

The piggyBac transposon has been long used to integrate foreign DNA into insect genomes.However,undesirable transgene expression can result from random insertions into the genome.In this study,the efficiency of chimeric Gal4-piggyBac transposase in directing integration onto a DNA target plasmid was evaluated in cultured silkworm Bombyx mori Bm-12 and fruit fly Drosophila Schneider 2(S2) cells.The Gal4-piggyBac transposase has a Gal4 DNA-binding domain(DBD),and the target plasmid has upstream activating sequences(UAS) to which the Gal4 DBD can bind with high affinity.The results indicate that,in the Bm-12 and S2 cells,transpositional activity of Gal4-piggyBac transposase was increased by 4.0 and 7.5 times,respectively,compared to controls,where Gal4-UAS interaction was absent.Moreover,the Gal4-piggyBac transposase had the ability of directing piggyBac element integration to certain sites of the target plasmid,although the target-directing specificity was not as high as expected.The chimeric piggyBac transposase has the potential for use in site-directed transgenesis and gene function research in B.mori.

Establishment of highly efficient transgenic system for black soldier fly(Hermetia illucens)

The black soldier fly(BSF),Hermetia illucens,is a promising insect for miti-gating solid waste problems as its larvae are able to bioconvert organic waste into valuable biomass.We recently reported a high-quality genome assembly of the BSF;analysis of this genome sequence will further the understanding of insect biology and identify genes that can be manipulated to improve efficiency of bioconversion.To enable genetic manip-ulation of the BSF,we have established the first transgenic methods for this economically important insect.We cloned and identified the ubiquitous actin5C promoter(Hiactin5C-p3k)and 3 endogenous U6 promoters(HiU6:1,HiU6:2,and HiU6:3).The Hiactin5C pro-moter was used to drive expression of a hyperactive variant of the piggyBac transposase,which exhibited up to 6-fold improvement in transformation rate when compared to the wild-type transposase.Furthermore,we evaluated the 3 HiU6 promoters using this trans-genic system.HiU6:1 and HiU6:2 promoters provided the highest knockdown efficiency with RNAi and are thus promising candidates for future Clustered Regularly Interspaced Short Palindromic Repeats(CRISPR)development.Overall,our findings provide valuable genetic engineering toolkits for basic research and genetic manipulation of the BSF.

Transgenic technologies in cassava for nutritional improvement and viral disease resistance: a key strategy for food security in Africa

As a major staple food source in Africa and other tropical developing countries, cassava(Manihot esculenta) provides basic sustenance for many subsistence farmers. However, cassava roots mainly accumulate starch with limited contribution of other nutrients such as proteins and vitamins. Also, two viral diseases, cassava mosaic disease(CMD) and cassava brown streak disease(CBSD), cause great losses in cassava production in subSaharan Africa and the Indian sub-continent. Genetic engineering provides promising approaches to improve nutritional value and increase resistance to viral diseases in cassava. This report presents several successful case studies on engineering protein content by overexpression of nutritious storage proteins and improving cassava resistance to viral diseases by RNA interference. Perspectives on the sustainable acquisition of new knowledge and development of biotechnology to solve these bottlenecks are discussed.