Green Fluorescent Protein Transgene Research Articles

Microvascular Fragment-Loaded Platelet-Rich Plasma Dressing Promotes Cutaneous Wound Healing.

Objective: Chronic wounds represent a considerable burden for the affected patients and the health care system. To overcome this problem, effective treatment strategies are urgently required. In this study, we tested a novel approach by combining platelet-rich plasma (PRP) and microvascular fragments (MVF) to create a prevascularized gel dressing. Approach: MVF were enzymatically isolated from the epididymal fat pads of transgenic green fluorescent protein (GFP)+ C57BL/6J donor mice. Subsequently, 5,000 MVF were suspended in 10 μL murine PRP as carrier and transferred into full-thickness skin wounds within dorsal skinfold chambers of C57BL/6J wild-type mice (PRP+MVF). Wound healing in comparison to empty wounds (control) and wounds filled with PRP alone was repeatedly analyzed throughout 14 days by means of stereomicroscopy, histology, and immunohistochemistry. Results: Planimetric assessment of the wound size over time revealed a significantly accelerated and improved healing of PRP+MVF-treated wounds when compared with PRP-treated and empty control wounds. These wounds also exhibited a significantly higher density of blood and lymph vessels, which originated from the GFP+ MVF isolates and effectively promoted granulation tissue formation inside the skin defects. Innovation: This study is the first to combine PRP and MVF for the improvement of wound healing. Conclusion: The combination of PRP and MVF represents a promising approach for the future treatment of wounds that do not heal spontaneously due to poor wound-healing conditions.

Recombinant AAV genome size effect on viral vector production, purification, and thermostability

Adeno-associated virus (AAV) has shown great promise as a viral vector for gene therapy in clinical applications. This work studied the effect of the genome size on AAV production, purification, and thermostability by producing AAV2-GFP using suspension adapted HEK293 cells via triple transfection using AAV plasmids containing the same green fluorescent protein (GFP) transgene with DNA stuffers for variable size AAV genomes consisting of 1.9, 3.4, and 4.9 kb (ITR to ITR). Production was performed at the small and large shake flask scales and the results showed that the 4.9 kb GFP genome had significantly reduced encapsidation compared to other genomes. The large shake flask productions were purified by AEX chromatography and the results suggest that the triple transfection condition significantly impacts the AEX retention time and resolution between the full and empty capsid peaks. Charge detection mass spectrometry (CD-MS) was performed on all AEX full capsid peak samples showing a wide distribution of empty, partial, full length, and co-packaged DNA in the capsids. The AEX purified samples were then analyzed by differential scanning fluorimetry (DSF) and the results suggest that sample formulation may improve the thermostability of AAV genome ejection melting temperature regardless of the packaged genome content.

Toxicity of Three Optical Brighteners: Potential Pharmacological Targets and Effects on Caenorhabditis elegans.

Optical brighteners (OBs) have become an integral part of our daily lives and culture, with a growing number of applications in various fields. Most industrially produced OBs are derived from stilbene, which has been found in environmental matrices. The main objectives for this work are as follows: first, to identify protein targets for DAST, FB-28, and FB-71, and second, to assess their effects in some behaviors physiologic of Caenorhabditis elegans. To achieve the first objective, each OB was tested against a total of 844 human proteins through molecular docking using AutoDock Vina, and affinities were employed as the main criteria to identify potential target proteins for the OB. Molecular dynamics simulations took and validated the best 25 docking results from two protein databases. The highest affinity was obtained for the Hsp70-1/DAST, CD40 ligand/FB-71, and CD40 ligand/FB-28 complexes. The possible toxic effects that OBs could cause were evaluated using the nematode C. elegans. The lethality, body length, locomotion, and reproduction were investigated in larval stage L1 or L4 of the wild-type strain N2. In addition, transgenic green fluorescent protein (GFP) strains were employed to estimate changes in relative gene expression. The effects on the inhibition of growth, locomotion, and reproduction of C. elegans nematodes exposed to DAST, FB-71, and FB-28 OBs were more noticeable with respect to lethality. Moreover, an interesting aspect in OB was increased the expression of gpx-4 and sod-4 genes associated with oxidative stress indicating a toxic response related to the generation of reactive oxygen species (ROS). In all cases, a clear concentration-response relationship was observed. It is of special attention that the use of OBs is increasing, and their different sources, such as detergents, textiles, plastics, and paper products, must also be investigated to characterize the primary emissions of OBs to the environment and to develop an adequate regulatory framework.

Enhancement of adeno-associated virus serotype 6 transduction into T cells with cell-penetrating peptides.

Adeno-associated viruses (AAVs) are gaining interest in the development of cellular immunotherapy. Compared to other viral vectors, AAVs can reduce the risk of insertional oncogenesis. AAV serotype 6 (AAV6) shows the highest efficiency for transducing T cells. Nevertheless, a multiplicity of infection (MOI) of up to one million viral genomes per cell is required to transduce the target cells effectively. Cell-penetrating peptides (CPPs) are short, positively charged peptides that easily translocate the plasma membranes and can facilitate the cellular uptake of a wide variety of cargoes, including small molecules, nucleic acids, drugs, proteins and viral vectors. The present study evaluated five CPPs (Antp, TAT-HA2, LAH4, TAT1 and TAT2) on their effects on enhancing transduction of AAV6 packaging a green fluorescent protein transgene into Jurkat T cell line. Vector incubation with peptides TAT-HA2 and LAH4 at a final concentration of 0.2mm resulted in an approximately two-fold increase in transduced cells. At the lowest MOI tested (1.25 × 104 ), using LAH4 resulted in a 10-fold increase in transduction efficiency. The peptide LAH4 increased the uptake of AAV6 viral particles in both Jurkat cells and mouse primary T cells. Regardless of the large size of the AAV6-LAH4 complexes, their internalization does not appear to depend on macropinocytosis. Overall, the present study reports an approach to significantly improve the delivery of transgenes into T cells using AAV6 vectors. Notably, the peptides TAT-HA2 and LAH4 contribute to improving the use of AAV6 as a gene delivery vector for the engineering of T cells.

Intraocular Axon Regeneration in a Model of Penetrating Eye Injury.

Purpose: Penetrating eye injuries commonly cause permanent loss of vision in patients. Unlike mammals, zebrafish can regenerate both damaged tissue and severed axons in the central nervous system. Here, we present a tractable adult zebrafish model to study intraocular axon regeneration after penetrating eye injury. Methods: To create consistent penetrating intraocular injuries, pins of standardized diameters were inserted into the eye through the cornea and penetrating the retina but not the underlying sclera. Transgenic gap43:GFP reporter fish were used to preferentially label retinal ganglion cells (RGCs) that respond to injury with regenerating axons. Retinas were fixed and flat mounted at various times postinjury to examine injury size, number of green fluorescent protein (GFP)-positive cells and axons, axonal varicosities, and rate of regeneration to the optic nerve head. Intraocular injection of colchicine was used to inhibit axon outgrow as a proof of principle that this method can be used to screen effects of pharmacological agents on intraocular axon regeneration. Results: Penetrating injury to the zebrafish retina results in robust axon regeneration by RGCs around and beyond the site of injury. The gap43:GFP transgene allows visualization of individual or small bundles of axons with varicosities and growth cones easily observable. Regeneration proceeded with most, if not all, axons reaching the optic nerve head by 3-day postinjury. A single intraocular injection of colchicine a day after injury was sufficient to delay axon regeneration at 2-days postinjury. Surprisingly, we identified a stereotypically located population of circumferential projecting neurons within the retina that upregulate gap43:GFP expression after injury. Conclusions: Penetrating injury to the adult gap43:GFP transgenic zebrafish eye is a model of successful intraocular axon regeneration. The pharmacological and genetic tools available for this organism should make it a powerful tool for dissecting the cellular, molecular, and genetic mechanisms of axon regeneration in the intraocular environment.

Scalable manufacturing of gene-modified human mesenchymal stromal cells with microcarriers in spinner flasks

Due to their immunomodulatory properties and in vitro differentiation ability, human mesenchymal stromal cells (hMSCs) have been investigated in more than 1000 clinical trials over the last decade. Multiple studies that have explored the development of gene-modified hMSC-based products are now reaching early stages of clinical trial programmes. From an engineering perspective, the challenge lies in developing manufacturing methods capable of producing sufficient doses of ex vivo gene-modified hMSCs for clinical applications. This work demonstrates, for the first time, a scalable manufacturing process using a microcarrier-bioreactor system for the expansion of gene-modified hMSCs. Upon isolation, umbilical cord tissue mesenchymal stromal cells (UCT-hMSCs) were transduced using a lentiviral vector (LV) with green fluorescent protein (GFP) or vascular endothelial growth factor (VEGF) transgenes. The cells were then seeded in 100 mL spinner flasks using Spherecol microcarriers and expanded for seven days. After six days in culture, both non-transduced and transduced cell populations attained comparable maximum cell concentrations (≈1.8 × 105 cell/mL). Analysis of the culture supernatant identified that glucose was fully depleted after day five across the cell populations. Lactate concentrations observed throughout the culture reached a maximum of 7.5 mM on day seven. Immunophenotype analysis revealed that the transduction followed by an expansion step was not responsible for the downregulation of the cell surface receptors used to identify hMSCs. The levels of CD73, CD90, and CD105 expressing cells were above 90% for the non-transduced and transduced cells. In addition, the expression of negative markers (CD11b, CD19, CD34, CD45, and HLA-DR) was also shown to be below 5%, which is aligned with the criteria established for hMSCs by the International Society for Cell and Gene Therapy (ISCT). This work provides a foundation for the scalable manufacturing of gene-modified hMSCs which will overcome a significant translational and commercial bottleneck.Key points• hMSCs were successfully transduced by lentiviral vectors carrying two different transgenes: GFP and VEGF• Transduced hMSCs were successfully expanded on microcarriers using spinner flasks during a period of 7 days• The genetic modification step did not cause any detrimental impact on the hMSC immunophenotype characteristics

Production of chickens with GFP-knockin in the Z chromosome and detection of GFP-positive chicks in the embryonic stage.

The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system, which is the most efficient and reliable tool for precisely targeted modification of the genome of living cells, has generated considerable excitement for industrial applications as well as scientific research. In this study, we developed a gene-editing and detection system for chick embryo sexing during the embryonic stage. By combining the CRISPR/Cas9 technical platform and germ cell-mediated germline transmission, we not only generated Z chromosome-targeted knockin chickens but also developed a detection system for fluorescence-positive male chicks in the embryonic stage. We targeted a green fluorescent protein (GFP) transgene into a specific locus on the Z chromosome of chicken PGCs, resulting in the production of ZGFP-knockin chickens. By mating ZGFP-knockin females (ZGFP/W) with wild males (Z/Z) and using a GFP detection system, we could identify chick sex, as the GFP transgene was expressed on the Z chromosome only in male offspring (ZGFP/Z) even before hatching. Our results demonstrate that the CRISPR/Cas9 technical platform with chicken PGCs facilitates the production of specific genome-edited chickens for basic research as well as practical applications.

Establishment of Transcriptional Gene Silencing Targeting the Promoter Regions of GFP, PDS, and PSY Genes in Cotton using Virus-Induced Gene Silencing.

Virus-induced gene silencing (VIGS) by deploying viral-based vectors such as tobacco rattle virus (TRV) is a homology-based gene silencing technique in post-transcriptional gene silencing (PTGS) and transcriptional gene silencing (TGS) to validate the function of particular genes. The study presented here showed the induction of DNA methylation in the promoter regions of three phenotypic marker genes in different cotton accessions, including two endogenous genes such as phytoene desaturase (PDS) and phytoene synthase (PSY), and an exogenous gene, such as green fluorescent protein (GFP). First, DNA methylation was established in transgenic GFP cotton where methylation persisted up to S3 generation. Afterward, the promoter of PSY was targeted following the same conditions. Significant silencing of PSY was observed and methylation of the promoter was found up to S2 generation in red leaf cotton as detected in GFP cotton. Silencing of PDS resulted in a photobleaching phenotype; interestingly, the strength of this phenotype was diverse within the plants and was not observed in the next generation. Bisulfite sequencing results showed methylation percentage of the cytosine residues was high at CG and CHG sites of the targeted promoter sequences in the silenced plants. The findings of this paper suggest that TRV-based vector system can be used to monitor DNA methylation for both exogenous and endogenous gene levels in cotton and offer a very useful tool for plant epigenetic modification.

WRKY29 transcription factor regulates ethylene biosynthesis and response in arabidopsis

The gaseous phytohormone ethylene participates in a lot of physiological processes in plants. 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (ACS, EC 4.4.1.14) and the ACC oxidase (ACO, EC 1.14.17.4) are key enzymes in ethylene biosynthesis. However, how ACSs and ACOs are regulated at the transcriptional level is largely unknown. In the present study, we showed that an Arabidopsis (Arabidopsis thaliana) WRKY-type transcription factor (TF), WRKY29 positively regulated the expression of ACS5, ACS6, ACS8, ACS11 and ACO5 genes and thus promoted basal ethylene production. WRKY29 protein was localized in nuclei and was a transcriptional activator. Overexpression of WRKY29 caused pleiotropic effect on plant growth, development and showed obvious response even without ACC treatment. Inducible overexpression of WRKY29 also reduced primary root elongation and lateral root growth. A triple response assay of overexpression and mutant seedlings of WRKY29 showed that overexpression seedlings had shorter hypocotyls than the transgenic GFP (Green Fluorescence Protein) control, while mutants had no difference from wild-type. A qRT-PCR assay demonstrated that expression of multiple ACSs and ACO5 was up-regulated in WRKY29 overexpression plants. A transactivation assay through dual luciferase reporter system confirmed the regulation of promoters of ACS5, ACS6, ACS8, ACS11 and ACO5 by WRKY29. Both in vivo chromatin immunoprecipitation (ChIP)- quantitative PCR and in vitro electrophoretic mobility shift assay (EMSA) revealed that WRKY29 directly bound to the promoter regions of its target genes. Taken together, these results suggest that WRKY29 is a novel TF positively regulating ethylene production by modulating the expression of ACS and ACO genes.

Magnetofection approach for the transformation of okra using green iron nanoparticles

Climate change, pesticide resistance, and the need for developing new plant varieties have galvanized biotechnologists to find new solutions in order to produce transgenic plants. Over the last decade scientists are working on green metallic nanoparticles to develop DNA delivery systems for plants. In the current study, green Iron nanoparticles were synthesized using leaf extract of Camellia sinensis (green tea) and Iron Chloride (FeCl3), the characterization and Confirmation was done using UV–VIS Spectroscopy, FTIR, SEM, and TEM. Using these nanoparticles, a novel method of gene transformation in okra plants was developed, with a combination of different Magnetofection factors. Maximum gene transformation efficiency was observed at the DNA to Iron-nanoparticles ratio of 1:20, by rotation of mixture (Plasmid DNA, Iron-nanoparticles, and seed embryo) at 800 rpm for 5 h. Using this approach, the transformation of the GFP (green fluorescent protein) gene was successfully carried out in Abelmoschus esculentus (Okra plant). The DNA transformation was confirmed by observing the expression of transgene GFP via Laser Scanning Confocal Microscope (LSCM) and PCR. This method is highly economical, adaptable, genotype independent, eco-friendly, and time-saving as well. We infer that this approach can be a potential solution to combat the yield and immunity challenges of plants against pathogens.

Cloning and base editing of GFP transgenic rhesus monkey and off-target analysis.

We report the cloning of a 12-year-old transgenic green fluorescent protein (GFP) monkey by somatic cell nuclear transfer (SCNT) and base editing of the embryos, accompanied with safety evaluation of adenine base editors (ABEs). We first show the ability of ABEmax to silence GFP through A-to-G editing of the GFP sequence in 293T cells. Subsequently, using donor cells from a monkey expressing GFP, we have successfully generated 207 ABEmax-edited (SCNT-ABE) and 87 wild-type (SCNT) embryos for embryo transfer, genotyping, and genome and transcriptome analysis. SCNT-ABE and SCNT embryos are compared for off-target analysis without the interference of genetic variants using a new method named as OA-SCNT. ABEmax does not induce obvious off-target DNA mutations but induces widespread off-target RNA mutations, 35% of which are exonic, in edited monkey embryos. These results provide important references for clinical application of ABE.

The tomato yellow leaf curl virus C4 protein alters the expression of plant developmental genes correlating to leaf upward cupping phenotype in tomato.

Tomato yellow leaf curl virus (TYLCV), a monopartite begomovirus in the family Geminiviridae, is efficiently transmitted by the whitefly, Bemisia tabaci, and causes serious economic losses to tomato crops around the world. TYLCV-infected tomato plants develop distinctive symptoms of yellowing and leaf upward cupping. In recent years, excellent progress has been made in the characterization of TYLCV C4 protein function as a pathogenicity determinant in experimental plants, including Nicotiana benthamiana and Arabidopsis thaliana. However, the molecular mechanism leading to disease symptom development in the natural host plant, tomato, has yet to be characterized. The aim of the current study was to generate transgenic tomato plants expressing the TYLCV C4 gene and evaluate differential gene expression through comparative transcriptome analysis between the transgenic C4 plants and the transgenic green fluorescent protein (Gfp) gene control plants. Transgenic tomato plants expressing TYLCV C4 developed phenotypes, including leaf upward cupping and yellowing, that are similar to the disease symptoms expressed on tomato plants infected with TYLCV. In a total of 241 differentially expressed genes identified in the transcriptome analysis, a series of plant development-related genes, including transcription factors, glutaredoxins, protein kinases, R-genes and microRNA target genes, were significantly altered. These results provide further evidence to support the important function of the C4 protein in begomovirus pathogenicity. These transgenic tomato plants could serve as basic genetic materials for further characterization of plant receptors that are interacting with the TYLCV C4.

Differential gene expression by RNA-seq during Alzheimer’s disease-like progression in the Drosophila melanogaster model

Alzheimer's disease (AD) is characterized by progressive, irreversible loss of memory and cognitive function. Drosophila melanogaster and other animal models are used to study several diseases, in order to elucidate unknown mechanisms and develop potential therapies. Molecular studies require biological samples and, for neuropathologies such as AD biopsy of the human brain, are invasive and potentially damaging. The solution is to use animal models, such as D. melanogaster, which is a model organism that can replace mammalian organisms in such studies. In this study, we evaluated the climbing ability and differential gene expression during AD progression due to the amylodoigenic pathway using RNA-seq, and we performed an in silico analysis of a fruit fly AD-like GFP (Green Fluorescent Protein) model with GFP expression in the pan-neural elav driver. A total of 1388 genes were differentially expressed in all analyzed groups. The main pathways related to those Differentially Expressed Genes (DEGs) during aging and AD progression were evaluated using the fly genes and human orthologs, in order to link genomic information to higher-order functional information with gene pathway mapping. We identified pathways present in all analyzed groups, such as metabolic pathways, ribosomal pathways, proteasome pathways and immune system pathways. Some of the genes were validated by qPCR. Knockdown of CG17754 gene by RNAi promoted degeneration in the fly eye, validating these findings in vivo. The identification of similarities in molecular pathways between the transgenic fly AD-like GFP model and mammals related to AD provides new insights into the use of this fly in screening novel anti-AD drugs.

High-pressure sprayed siRNAs influence the efficiency but not the profile of transitive silencing.

In plants, small interfering RNAs (siRNAs) are a quintessential class of RNA interference (RNAi)-inducing molecules produced by the endonucleolytic cleavage of double-stranded RNAs (dsRNAs). In order to ensure robust RNAi, siRNAs are amplified through a positive feedback mechanism called transitivity. Transitivity relies on RNA-DIRECTED RNA POLYMERASE 6 (RDR6)-mediated dsRNA synthesis using siRNA-targeted RNA. The newly synthesized dsRNA is subsequently cleaved into secondary siRNAs by DICER-LIKE (DCL) endonucleases. Just like primary siRNAs, secondary siRNAs are also loaded into ARGONAUTE proteins (AGOs) to form an RNA-induced silencing complex reinforcing the cleavage of the target RNA. Although the molecular players underlying transitivity are well established, the mode of action of transitivity remains elusive. In this study, we investigated the influence of primary target sites on transgene silencing and transitivity using the green fluorescent protein (GFP)-expressing Nicotiana benthamiana 16C line, high-pressure spraying protocol, and synthetic 22-nucleotide (nt) long siRNAs. We found that the 22-nt siRNA targeting the 3' of the GFP transgene was less efficient in inducing silencing when compared with the siRNAs targeting the 5' and middle region of the GFP. Moreover, sRNA sequencing of locally silenced leaves showed that the amount but not the profile of secondary RNAs is shaped by the occupancy of the primary siRNA triggers on the target RNA. Our findings suggest that RDR6-mediated dsRNA synthesis is not primed by primary siRNAs and that dsRNA synthesis appears to be generally initiated at the 3'-end of the target RNA.

Targeted mutagenesis in plants using Beet curly top virus for efficient delivery of CRISPR/Cas12a components

Genome editing using CRISPR/Cas is rapidly being developed for gene targeting in eukaryotes including plants. However, gene targeting by homology-directed DNA recombination (HDR) is an infrequent event compared to the dominant DNA repair by non-homologous end-joining. Another bottleneck is the ineffective delivery of CRISPR/Cas components into plant cells. To overcome these constraints, here a geminiviral replicon from Beet curly top virus (BCTV) has been produced with a wide host range and high DNA accumulation capacity for efficient delivery of CRISPR/Cas12a components into plant cells. Initially, a BCTV replicon was prepared after removing the virion sense genes from an infectious full-length clone for agrobacterium mediated infection. This replicon expressed a green fluorescent protein (GFP) marker gene at a high level compared to T-DNA binary vector. In transient assay, the BCTV replicon produced a higher rate of mutagenesis and HDR in the GFP transgene in Nicotiana benthamiana through efficient delivery of CRISPR/Cas12a components compared to the cognate T-DNA control. This was through a range of complete or partial HDR for conversion of GFP into YFP after exchange of a single amino acid (Thr224Tyr) in the target gene. In addition, induced mutagenesis and HDR in the target gene were heritable. Thus, the BCTV replicon provides a new tool for efficient delivery of CRISPR/Cas12a components that could be used in a wide range of dicotyledonous plants. The established GFP to YFP system and the GFP mutant line produced also enable further optimization and understanding of HDR in plants via CRISPR/Cas12a system using geminiviral replicons.

Rat perichondrium transplanted to articular cartilage defects forms articular-like, hyaline cartilage.

Perichondrium autotransplants have been used to reconstruct articular surfaces destroyed by infection or trauma. However, the role of the transplanted perichondrium in the healing of resurfaced joints has not been investigated. Perichondrial and periosteal tissues were harvested from rats hemizygous for a ubiquitously expressed enhanced green fluorescent protein (EGFP) transgene and transplanted into full-thickness articular cartilage defects at the trochlear groove of distal femur in wild-type littermates. As an additional control, cartilage defects were left without a transplant (no transplant control). Distal femurs were collected 3, 14, 56, 112days after surgery. Tracing of transplanted cells showed that both perichondrium and periosteum transplant-derived cells made up the large majority of the cells in the regenerated joint surfaces. Perichondrium transplants contained SOX9 positive cells and with time differentiated into a hyaline cartilage that expanded and filled out the defects with Col2a1-positive and Col1a1-negative chondrocytes and a matrix rich in proteoglycans. At later timepoints the cartilaginous perichondrium transplants were actively remodeled into bone at the transplant-bone interface and at post-surgery day 112 EGFP-positive perichondrium cells at the articular surface were positive for Prg4. Periosteum transplants initially lacked SOX9 expression and despite a transient increase in SOX9 expression and chondrogenic differentiation, remained Col1a1 positive, and were continuously thinning as periosteum-derived cells were incorporated into the subchondral compartment. Perichondrium and periosteum transplanted to articular cartilage defects did not just stimulate regeneration but were themselves transformed into cartilaginous articular surfaces. Perichondrium transplants developed into an articular-like, hyaline cartilage, whereas periosteum transplants appeared to produce a less resilient fibro-cartilage.

Generation of the Y-chromosome linked red fluorescent protein transgenic mouse model and sexing at the preimplantation stage.

In mammals, sexual fate is determined by the chromosomes of the male and female gametes during fertilization. Males (XY) or females (XX) are produced when a sperm containing a Y or X-chromosome respectively fertilizes an X-chromosome-containing unfertilized egg. However, sexing of preimplantation stage embryos cannot be conducted visually. To address this, transgenic male mouse models with the ubiquitously expressed green fluorescent protein (GFP) transgene on X- (X-GFP) or Y-chromosomes (Y-GFP) have been established. However, when crossed with wild-type females, sexing of the preimplantation stage embryos by observing the GFP signal is problematic in some cases due to X-inactivation, loss of Y-chromosome (LOY), or loss of transgene fluorescence. In this study, a mouse model with the ubiquitously expressed red fluorescent protein (RFP) transgene on the Y-chromosome was generated since RFP is easily distinguishable from GFP signals. Unfortunately, the ubiquitously expressed tdTomato RFP transgene on the Y-chromosome (Y-RFP) mouse showed the lethal phenotype after birth. No lethal phenotypes were observed when the mitochondrial locating signal N-terminal of tdTomato (mtRFP) was included in the transgene construct. Almost half of the collected fertilized eggs from Y-mtRFP male mice crossed with wild-type females had an RFP signal at the preimplantation stage (E1.5). Therefore, XY eggs were recognized as RFP-positive embryos at the preimplantation stage. Furthermore, 100% sexing was observed at the preimplantation stage using the X-linked GFP/Y-linked RFP male mouse. The established Y-mtRFP mouse models may be used to study sex chromosome related research.

Topically delivered 22 nt siRNAs enhance RNAi silencing of endogenous genes in two species

Main conclusion22 nt siRNAs applied to leaves induce production of transitive sRNAs for targeted genes and can enhance local silencing. Systemic silencing was only observed for a GFP transgene.RNA interference (RNAi) is a gene silencing mechanism important in regulating gene expression during plant development, response to the environment and defense. Better understanding of the molecular mechanisms of this pathway may lead to future strategies to improve crop traits of value. An abrasion method to deliver siRNAs into leaf cells of intact plants was used to investigate the activities of 21 and 22 nt siRNAs in silencing genes in Nicotiana benthamiana and Amaranthus cruentus. We confirmed that both 21 and 22 nt siRNAs were able to silence a green fluorescent protein (GFP) transgene in treated leaves of N. benthamiana, but systemic silencing of GFP occurred only when the guide strand contained 22 nt. Silencing in the treated leaves of N. benthamiana was demonstrated for three endogenous genes: magnesium cheletase subunit I (CHL-I), magnesium cheletase subunit H (CHL-H), and GENOMES UNCOUPLED4 (GUN4). However, systemic silencing of these endogenous genes was not observed. Very high levels of transitive siRNAs were produced for GFP in response to treatment with 22 nt siRNAs but only low levels were produced in response to a 21 nt siRNA. The endogenous genes tested also produced transitive siRNAs in response to 22 nt siRNAs. 22 nt siRNAs produced greater local silencing phenotypes than 21 nt siRNAs for three of the genes. These special properties of 22 nt siRNAs were also observed for the CHL-H gene in A. cruentus. These experiments suggest a functional role for transitive siRNAs in amplifying the RNAi response.

An N-ethyl-N-Nitrosourea Mutagenesis Screen in Mice Reveals a Mutation in Nuclear Respiratory Factor 1 (Nrf1) Altering the DNA Methylation State and Correct Embryonic Development.

Simple SummaryIn this work, we aimed to discover unknown genes that are important in the regulation of other genes. These genes often play an important role during the development of the embryo. By screening thousands of mice, we found a gene, namely, Nuclear Respiratory Factor 1 (Nrf1), that controls the switching on and off of other genes. Mice with a defective Nrf1 present lesser levels of the gene and embryonic delay. When the mutation is in both chains of the DNA, mice are not born and die in the uterus. Our work unveils a novel, previously unknown functionality of Nrf1 and provides a new mice model for the study of diseases caused by a defective Nrf1.We have established a genome-wide N-ethyl-N-nitrosourea (ENU) mutagenesis screen to identify novel genes playing a role in epigenetic regulation in mammals. We hypothesize that the ENU mutagenesis screen will lead to the discovery of unknown genes responsible of the maintenance of the epigenetic state as the genes found are modifiers of variegation of the transgene green fluorescent protein (GFP) expression in erythrocytes, which are named MommeD. Here we report the generation of a novel mutant mouse line, MommeD46, that carries a new missense mutation producing an amino acid transversion (L71P) in the dimerization domain of Nuclear Respiratory Factor 1 (Nrf1). The molecular characterization of the mutation reveals a decrease in the Nrf1 mRNA levels and a novel role of Nrf1 in the maintenance of the DNA hypomethylation in vivo. The heritability of the mutation is consistent with paternal imprinting and haploinsufficiency. Homozygous mutants display embryonic lethality at 14.5 days post-coitum and developmental delay. This work adds a new epi-regulatory role to Nrf1 and uncovers unknown phenotypical defects of the Nrf1 hypomorph. The generated mouse line represents a valuable resource for studying NRF1-related diseases.

Efficient genome editing and gene knockout in Setaria viridis with CRISPR/Cas9 directed gene editing by the non-homologous end-joining pathway.

The CRISPR/Cas9 system has been used for genome editing in several organisms, including higher plants. This system induces site-specific mutations in the genome based on the nucleotide sequence of engineered guide RNAs. The complex genomes of C4 grasses makes genome editing a challenge in key grass crops like maize (Zea mays), sorghum (Sorghum bicolor), Brachiaria spp., switchgrass (Panicum virgatum), and sugarcane (Saccharum spp.). Setaria viridis is a diploid C4 grass widely used as a model for these C4 crop plants. Here, an optimized CRISPR/Cas9 binary vector that exploits the non-homologous end joining (NHEJ) system was used to knockout a green fluorescent protein (gfp) transgene in S. viridis accession A10.1. Transformation of embryogenic callus by A. tumefaciens generated ten glufosinate-ammonium resistant transgenic events. In the T0 generation, 60% of the events were biallelic mutants in the gfp transgene with no detectable accumulation of GFP protein and without insertions or deletions in predicted off-target sites. The gfp mutations generated by CRISPR/Cas9 were stable and displayed Mendelian segregation in the T1 generation. Altogether, the system described here is a highly efficient genome editing system for S. viridis, an important model plant for functional genomics studies in C4 grasses. Also, this system is a potential tool for improvement of agronomic traits in C4 crop plants with complex genomes.