EGFP Gene Research Articles

CRISPR-Cas12a-based genome editing and transcriptional repression for biotin synthesis in Pseudomonas mutabilis.

To establish a dual-function clustered regularly interspaced short palindromic repeats (CRISPR)-Cas12a system combined genome editing and transcriptional repression for multiplex metabolic engineering of Pseudomonas mutabilis. This CRISPR-Cas12a system consisted of two plasmids that enabled single gene deletion, replacement, and inactivation with efficiency >90% for most targets within 5 days. With the guidance of truncated crRNA containing 16 bp spacer sequences, a catalytically active Cas12a could be employed to repress the expression of the reporter gene eGFP up to 66.6%. When bdhA deletion and eGFP repression were tested simultaneously by transforming a single crRNA plasmid and Cas12a plasmid, the knockout efficiency reached 77.8% and the expression of eGFP was decreased by >50%. Finally, the dual-functional system was demonstrated to increase the production of biotin by 3.84-fold, with yigM deletion and birA repression achieved simultaneously. This CRISPR-Cas12a system is an efficient genome editing and regulation tool to facilitate the construction of P. mutabilis cell factories.

Effect of temperature and IRF-9 gene-knockout on dynamics of vRNA, cRNA, and mRNA of viral hemorrhagic septicemia virus (VHSV)

The replication of viral hemorrhagic septicemia virus (VHSV) in appropriate host cells depends on environmental factors and the host cell's immunity. The dynamics of each VHSV RNA strand (vRNA, cRNA, and mRNA) in different conditions can provide a clue on the viral replication strategies, which can be a base for the development of efficient control measures. As VHSV is known to be sensitive to temperature and type I interferon (IFN) responses, in this study, we analyzed the effect of temperature difference (15 °C and 20 °C) and IRF-9 gene knockout on the dynamics of the three VHSV RNA strands in Epithelioma papulosum cyprini (EPC) cells using a strand-specific RT-qPCR. The tagged primers designed in this study successfully worked to quantify the three strands of VHSV. In the results of the temperature effect, the higher speed in viral mRNA transcription and the significantly higher (more than 10 times at 12–36 h) copy number of cRNA at 20 °C compared to those at 15 °C suggested the positive effect of high temperature on VHSV replication. In the results of the IRF-9 gene knockout effect, although IRF-9 gene knockout did not bring a dramatic effect on VHSV replication compared to the temperature effect, the increase of mRNA in IRF-9 KO cells was faster than normal EPC cells, which was reflected in the copy numbers of cRNA and vRNA. The IRF-9 gene knockout effect was not dramatic even in the replication of rVHSV-ΔNV-eGFP that harbors eGFP gene ORF instead of NV gene ORF. These results suggest that VHSV may be highly susceptible to pre-activated type I IFN responses but not highly susceptible to post-infection-mediated type I IFN responses or lowered type I IFN before infection. In both experiments of temperature effect and IRF-9 gene knockout effect, the copy number of cRNA never exceeded the copy number of vRNA at all assay times, suggesting that the binding efficiency of the RNP complex to the 3′ end of cRNA might be lower than that to the 3′ end of vRNA. Further research is needed to elucidate the regulatory mechanism that limits the amount of cRNA at an appropriate level during VHSV replication.

Random Integration Analysis of Recombinant Adeno-Associated Virus 6 Packaged in Sf9 Insect Cells

Recently, there have been growing concerns over the integration of recombinant adeno-associated virus (rAAV) used in gene therapy. Wild-type adeno-associated virus (AAV) site specifically integrates into AAVS1 site of human genome, while rAAV randomly integrates into host chromosomes at low frequencies. This research aims to study the random integration events of rAAV6-EGFP packaged in Sf9 insect cells. Baculo-Sf9 manufacturing platform has the advantages of high-density suspension culture of Sf9 insect cells and large-scale production of rAAV vectors. In this study, we used different doses of Baculo-Sf9 produced rAAV6-EGFP to transduce HEK293T cells and A549-implanted tumors in vitro and in vivo. Using flow cytometry and fluorescence microscopy, we studied their EGFP gene expression efficiencies and EGFP fluorescence intensities. Using inverse nested PCR and DNA sequencing, random integration sites of rAAV6-EGFP genome into human chromosomes were identified. In vitro results showed that gene expression efficiencies became stable after 20 days and random integration frequencies were 0.2-4.2%. Both in vitro and in vivo results indicated that random integration of Baculo-Sf9 rAAV6 was dose-dependent. Sequencing results showed two random integration sites, which were on human chromosomes 8 and 12. The findings suggest that we should use as low dose of rAAV vector as possible for safe gene therapy.

Self-assembled endogenous DNA nanoparticles for auto-release and expression of the eGFP gene in Bacillus subtilis

The development of DNA delivery techniques is critical to promote the wider use of deoxyribonucleic acids as cellular transporters. The present study aimed to develop a type of DNA nanoparticle (citZ-box) to automatically load and release cargo. The restriction enzyme can cleave citZ-boxes at pro-designed sites, and the enhanced green fluorescent protein gene (eGFP) can be delivered into the B. subtilis protoplasts by them. The process of eGFP expression is recorded using a confocal microscope over 4 h. Here, multiscaffold and multimodular designs are used for citZ-box assembly with a DAEDALUS module, DX_cage_design and rem (edge_length, 21), to ensure the structure was predicted as B-type DNA. Finally the citZ-box is estimated to be a 50.7 nm cube. The 3D structure of the citZ-box particle is detected to be approximately 50.3 ± 0.3 nm. DNA nanoparticles prepared as citZ-boxes have great potential as drug carriers with automatic loading and releasing abilities.

Deletion of gene OV132 attenuates Orf virus more effectively than gene OV112.

Orf virus (ORFV), a Parapoxvirus in Poxviridae, infects sheep and goats resulting in contagious pustular dermatitis. ORFV is regarded as a promising viral vector candidate for vaccine development and oncolytic virotherapy. Owing to their potential clinical application, safety concerns have become increasingly important. Deletion of either the OV132 (encoding vascular endothelial growth factor, VEGF) or OV112 (encoding the chemokine binding protein, CBP) genes reduced ORFV infectivity, which has been independently demonstrated in the NZ2 and NZ7 strains, respectively. This study revealed that the VEGF and CBP gene sequences of the local strain (TW/Hoping) shared a similarity of 47.01% with NZ2 and 90.56% with NZ7. Due to the high sequence divergence of these two immunoregulatory genes among orf viral strains, their contribution to the pathogenicity of Taiwanese ORFV isolates was comparatively characterized. Initially, two ORFV recombinants were generated, in which either the VEGF or CBP gene was deleted and replaced with the reporter gene EGFP. In vitro assays indicated that both the VEGF-deletion mutant ORFV-VEGFΔ-EGFP and the CBP deletion mutant ORFV-CBPΔ-EGFP were attenuated in cells. In particular, ORFV-VEGFΔ-EGFP significantly reduced plaque size and virus yield compared to ORFV-CBPΔ-EGFP and the wild-type control. Similarly, in vivo analysis revealed no virus yield in the goat skin biopsy infected by ORFV-VEGFΔ-EGFP, and significantly reduced the virus yield of ORFV-CBPΔ-EGFP relative to the wild-type control. These results confirmed the loss of virulence of both deletion mutants in the Hoping strain, whereas the VEGF-deletion mutant was more attenuated than the CBP deletion strain in both cell and goat models. KEY POINTS: • VEGF and CBP genes are crucial in ORFV pathogenesis in the TW/Hoping strain • The VEGF-deletion mutant virus was severely attenuated in both cell culture and animal models • Deletion mutant viruses are advantageous vectors for the development of vaccines and therapeutic regimens.

Study on the central neural pathway and the relationship between the heart and small intestine via a dual neural tracer.

Despite very different functions, studies increasingly report that there may be a potential central nervous anatomical connection between the heart and the small intestine. In this study, the central nervous anatomical relationship between the heart and small intestine was studied via a viral tracer. Pseudorabies virus (PRV) syngeneic strains with different fluorescent reporter genes (eGFP or mRFP) were microinjected into the heart walls and small intestinal walls of male C57BL/6J using glass microelectrode. The results showed that the co-labeled nuclei in the brain were lateral periaqueductal gray (LPAG) and ventrolateral periaqueductal gray (VLPAG) in the midbrain, mesencephalic trigeminal nucleus (Me5), and motor trigeminal nucleus anterior digastric Part (5Adi) in the pons. The co-labeled sites in the spinal cord were intermediolateral column (IML) in the second thoracic vertebra, IML and lamina 7 of the spinal gray (7SP) in the third thoracic vertebra, and IML in the fourth thoracic vertebra. Our data show that there is a neuroanatomical connection between the small intestine and the heart in the central nervous system (CNS). Neuroanatomical integration of the heart and small intestine may provide a basis for revealing the physiological and pathological interactions between the circulatory and digestive systems. The interactions may be mediated more effectively through sympathetic nerves.

Cytotoxic effect of cloned EGFP gene on NCI-H727 cell line via genetically engineered gene transfer system

Introduction and Aim: Cancers are a complex group of genetic illnesses that develop through multistep, mutagenic processes which can invade or spread throughout the body. Recent advances in cancer treatment involve oncolytic viruses to infect and destroy cancer cells. The Newcastle disease virus (NDV), an oncolytic virus has shown to have anti-cancer effects either directly by lysing cancer cells or indirectly by activating the immune system. The green fluorescent protein (GFP) has been widely used in studying the anti-tumor activity of oncolytic viruses. This study aimed to study the anticancer effect of a recombinant rNDV-GFP clone on NCI-H727 lung carcinoma cell line in vitro. Materials and Methods: The GFP gene was inserted to a NDV strain to create a recombinant NDV (rNDV- GFP) using reverse genetics technology. The MTT assay was used in evaluating the oncolytic effect of rNDV- GFP on the lung carcinoma NCI-H727 cells. Light and fluorescent microscopy was used to study the cytopathic effects of rNDV-GFP. Results: MTT assay showed that rNDV-GPF inhibited the NCI-H727 tumor cell death in a time-dependent manner. A significant inhibitory effect (78.3%) for rNDV-GPF on cancer cells was observed at 96h in comparison to rNDV (22.7%) and the cytotoxicity rate was directly proportional to the MOI used. Microscopic studies showed rNDV-GPF to induce cytopathic effect post 24 h of infection. Conclusion: The GFP-expressing recombinant NDV strains exhibited encouraging results in terms of tumor growth inhibition. Our research set the groundwork for employing recombinant NDV as an anticancer viral vector.

Mycobacterium leprae hsp18 promoter-EGFP transcriptional fusion construct: Environmental stress and strain-specific expression

The Hsp18 protein is a major T-cell antigen of Mycobacterium leprae belonging to the family of small heat-shock proteins. The protein is specifically regulated at post-translational level during the intracellular growth of M. leprae within macrophages due to auto-phosphorylation, indicating its importance in the survival of the bacterium. The promoter and regulatory sequences that control hsp18 expression are located within a 256-bp sequence upstream of the translation start site. However, there are no studies describing either characterization of the hsp18 promoter or its genetic regulation. Therefore, we constructed an hsp18-EGFP transcriptional fusion in an E. coli–Mycobacterium shuttle vector. A 168-bp sequence comprising the hsp18 promoter was cloned upstream of the EGFP gene and transformed in M. smegmatis, and the integration of the construct was confirmed by Southern hybridization. hsp18 promoter activity was measured by analyzing EGFP expression in M. smegmatis and Escherichia coli grown under different environmental stress conditions normally encountered by M. leprae in vivo. We found that the 168-bp upstream sequence of hsp18 could function as a promoter, and the regulation of hsp18 expression was host-, environmental stress-, and temperature-dependent. Appreciable EGFP expression was detected in M. smegmatis grown under normal conditions, and theexpression was significantly increased by environmental stress. However, EGFP expression was observed in E. coli only under stress conditions. Comparative sequence analysis revealed the putative sigma factor C (SigC)-binding site within the 168-bp promoter sequence of hsp18, which might be involved in the regulation of hsp18 expression during stress conditions in M. leprae. Thus, our data demonstrated the transcriptional regulation of hsp18 expression in response to different environmental stress conditions, possibly through SigC in Mycobacterium. Further, this shuttle vector could be used for the functional characterization of M. leprae genes in heterologous systems.

Development of rapid neutralization assay of viral hemorrhagic septicemia virus (VHSV) based on chimeric rhabdovirus expressing heterologous glycoprotein

The titer of neutralizing antibodies (NAbs) against viral hemorrhagic septicemia virus (VHSV) has been determined by conventional neutralization assay based on the observation of cytopathic effect (CPE) and plaque formation in cultured cells. However, this method requires several days for the determination and can be affected by operator bias. To develop a rapid and high-throughput neutralization assay against VHSV, we rescued a surrogate chimeric snakehead rhabdovirus, rSHRV-Gvhsv-eGFP, which has the enhanced green fluorescent protein (eGFP) gene between N and P genes and has VHSV G gene instead of SHRV G gene in the genome. The efficacy of rSHRV-Gvhsv-eGFP to determine serum neutralization activity was evaluated using various serum samples derived from New Zealand white rabbits and olive flounder (Paralichthys oliavaceus). Although neutralization titers analyzed using rSHRV-Gvhsv-eGFP were similar to the titers measured using rVHSV-A-eGFP, the time needed for the determination of neutralization titer was much shortened (24 h for rSHRV-Gvhsv-eGFP and 48 h for rVHSV-A-eGFP), proving the usefulness of rSHRV-Gvhsv-eGFP for the neutralization assay against VHSV. In addition, as the neutralization activities using rSHRV-Gvhsv-eGFP could be well-observed without adding fresh serum as a complement source, no preparation is required for the optimization of control fresh serum from naïve fish. The present results suggest that the rapid neutralization assay using rSHRV-Gvhsv-eGFP can be used to investigate neutralization activities against VHSV.

Antibiotic-Free Nanoplasmids as Promising Alternatives for Conventional DNA Vectors.

DNA vaccines with their extraordinary properties are the best choice as vectors for subunit vaccines but are not in compliance with safety regulations, mainly because of the antibiotic resistance genes on their backbone. New generations of plasmids with minimum bacterial backbones are now developed as promising alternatives to pass the safety rules and be replaced for conventional plasmids. Here we have compared the nanoplasmid (with RNA-out selection system and professional HTLV-1 containing promoter) and the conventionally used pcDNA plasmid, as regards the transfection efficiency. The EGFP gene was cloned in both pcDNA-3.1+ and NTC9385R-MSC and transfected into COS-7 cells for expression evaluation by flow cytometry. Meanwhile, qPCR was used to analyze the EGFP mRNA copy numbers. It was concluded that the nanoplasmid, with its extraordinary properties, can be a tempting alternative to conventional pcDNA in equal or equimolar concentrations for vaccine design. These promising results can put DNA vaccines back into focus, especially regarding diseases controlled by robust cellular immune responses.

Enhancing stability of recombinant CHO cells by CRISPR/Cas9-mediated site-specific integration into regions with distinct histone modifications.

Chinese hamster ovary (CHO) cells are the most important platform for producing biotherapeutics. Random integration of a transgene into epigenetically instable regions of the genome results in silencing of the gene of interest and loss of productivity during upstream processing. Therefore, cost- and time-intensive long-term stability studies must be performed. Site-specific integration into safe harbors is a strategy to overcome these limitations of conventional cell line design. Recent publications predict safe harbors in CHO cells based on omics data sets or by learning from random integrations, but those predictions remain theory. In this study, we established a CRISPR/Cas9-mediated site-specific integration strategy based on ChIP-seq data to improve stability of recombinant CHO cells. Therefore, a ChIP experiment from the exponential and stationary growth phase of a fed-batch cultivation of CHO-K1 cells yielded 709 potentially stable integration sites. The reporter gene eGFP was integrated into three regions harboring specific modifications by CRISPR/Cas9. Targeted Cas9 nanopore sequencing showed site-specific integration in all 3 cell pools with a specificity between 23 and 73%. Subsequently, the cells with the three different integration sites were compared with the randomly integrated donor vector in terms of transcript level, productivity, gene copy numbers and stability. All site-specific integrations showed an increase in productivity and transcript levels of up to 7.4-fold. In a long-term cultivation over 70 generations, two of the site-specific integrations showed a stable productivity (>70%) independent of selection pressure.

Isolation and evaluation of strong endogenous promoters for the heterologous expression of proteins in Pichia pastoris.

The heterologous expression of biosynthetic pathway genes for pharmaceutical or fine chemical production usually requires to express more than one gene in the host cells. In eukaryotes, the pathway flux is typically balanced by controlling the transcript levels of the genes involved. It is difficult to balance the stoichiometric fine-tuning of the reaction steps of the pathway by acting on one or two promoters. Furthermore, the promoter used should not be identical to avoid loss of inserted genes by recombination or dilute its transcription factors. Based on RNA-seq data, 18 candidate genes with the highest transcription levels at three carbon sources (glucose, glycerol and methanol) were selected and their promoter regions were isolated from GS115 genome. The performance of these promoters on the level of protein production was evaluated using LacZ and EGFP genes as the reporters, respectively. These isolated promoters all exhibited activity to express LacZ gene. Using LacZ as a reporter, of the 18 promoter candidates, 9 promoters showed higher expression levels for the reporter compare to pGAP, a strong promoter widely used for constitutive expression of heterologous proteins in Pichia pastoris. These promoters with high expression levels were further employed to evaluate secreted expression using EGFP as a reporter. 6 promoters exhibited stronger protein expression compare to pGAP. Interestingly, the protein expression driven by pFDH1 was slightly higher than that of commonly used pAOX1 at methanol, and methanol-induced expression of pFDH1 was not repressed by glycerol. The various promoters identified in this study could be used for heterologous expression of biosynthetic pathway genes for pharmaceutical or fine chemical production. the methanol-induced pFDH1 that is not repressed by glycerol is an attractive alternative to pAOX1 and may provide a novel way to produce heterologous proteins in Pichia pastoris.

Improving AOX1 promoter efficiency by overexpression of Mit1 transcription factor.

Reprogramming in transcriptional regulation provides an effective tool for adjusting cellular metabolic activities. The strong methanol-inducible alcohol oxidase-1 promoter (pAOX1) is commonly used for heterologous gene expression in the yeast Pichia pastoris. Here, we present a novel Pichia pastoris strain engineered to co-express methanol-induced transcription factor 1 (Mit1) and the target protein. Mit1 upregulates pAOX1 in response to methanol. Two model proteins (VEGF and eGFP) have been used as the target proteins under the control of pAOX1. The sequence of Mit1 had obtained from the yeast genome and likewise cloned under the control of pAOX1. The results indicated a 1.9 and 2.2 fold increase in the detected VEGF and eGFP, respectively, when co-expressed with Mit1. Furthermore, the double-recombinant cells, containing Mit-1 and eGFP, produced 1.3 fold more eGFP when the methanol feeding concentration was doubled. The real-time PCR indicated a slight increase in the Mit1 expression, probably due to the negative regulatory feedback loop that exists for the intrinsic yeast Mit1. Overexpression of Mit1 also led to duplication of AOX1 enzyme activity, which may enhance the yeast cells' capacity for methanol detoxification. Overexpression of Mit1 could be considered a promising strategy for upregulation of target recombinant proteins in Pichia pastoris. Intracellular overexpression of Mit1 upregulates the heterologous target gene (eGFP) production, which is expressed under the control of pAOX1.

Recombinant Adeno-Associated Virus-Mediated Editing of the G551D Cystic Fibrosis Transmembrane Conductance Regulator Mutation in Ferret Airway Basal Cells.

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene cause cystic fibrosis (CF), a chronic disease that affects multiple organs, including the lung. We developed a CF ferret model of a scarless G551→D substitution in CFTR (CFTRG551D-KI), enabling approaches to correct this gating mutation in CF airways via gene editing. Homology-directed repair (HDR) was tested in Cas9-expressing CF airway basal cells (Cas9-GKI) from this model, as well as reporter basal cells (Y66S-Cas9-GKI) that express an integrated nonfluorescent Y66S-EGFP (enhanced green fluorescent protein) mutant gene to facilitate rapid assessment of HDR by the restoration of fluorescence. Recombinant adeno-associated virus (rAAV) vectors were used to deliver two DNA templates and sgRNAs for dual-gene editing at the EGFP and CFTR genes, followed by fluorescence-activated cell sorting of EGFPY66S-corrected cells. When gene-edited airway basal cells were polarized at an air-liquid interface, unsorted and EGFPY66S-corrected sorted populations gave rise to 26.0% and 70.4% CFTR-mediated Cl- transport of that observed in non-CF cultures, respectively. The consequences of gene editing at the CFTRG551D locus by HDR and nonhomologous end joining (NHEJ) were assessed by targeted gene next-generation sequencing (NGS) against a specific amplicon. NGS revealed HDR corrections of 3.1% of G551 sequences in the unsorted population of rAAV-infected cells, and 18.4% in the EGFPY66S-corrected cells. However, the largest proportion of sequences had indels surrounding the CRISPR (clustered regularly interspaced short palindromic repeats) cut site, demonstrating that NHEJ was the dominant repair pathway. This approach to simultaneously coedit at two genomic loci using rAAV may have utility as a model system for optimizing gene-editing efficiencies in proliferating airway basal cells through the modulation of DNA repair pathways in favor of HDR.

CRISPR-Cas9-directed gene tagging using a single integrase-defective lentiviral vector carrying a transposase-based Cas9 off switch

Locus-directed DNA cleavage induced by the CRISPR-Cas9 system triggers DNA repair mechanisms allowing gene repair or targeted insertion of foreign DNA. For gene insertion to be successful, availability of a homologous donor template needs to be timed with cleavage of the DNA by the Cas9 endonuclease guided by a target-specific single guide RNA (sgRNA). We present a novel approach for targeted gene insertion based on a single integrase-defective lentiviral vector (IDLV) carrying a Cas9 off switch. Gene insertion using this approach benefits from transposon-based stable Cas9 expression, which is switched off by excision-only transposase protein co-delivered in IDLV particles carrying a combined sgRNA/donor vector. This one-vector approach supports potent (up to >80%) knockin of a full-length EGFP gene sequence. This traceless cell engineering method benefits from high stable levels of Cas9, timed intracellular availability of the molecular tools, and a built-in feature to turn off Cas9 expression after DNA cleavage. The simple technique is based on transduction with a single IDLV, which holds the capacity to transfer larger donor templates, allowing robust gene knockin or tagging of genes in a single step.

Efficient fluorescence-based localization technique for real-time tracking endophytes route in host-plants colonization.

Bacterial isolates that enhance plant growth and suppress plant pathogens growth are essential tools for reducing pesticide applications in plant production systems. The objectives of this study were to develop a reliable fluorescence‐based technique for labeling bacterial isolates selected as biological control agents (BCAs) to allow their direct tracking in the host‐plant interactions, understand the BCA localization within their host plants, and the route of plant colonization. Objectives were achieved by developing competent BCAs transformed with two plasmids, pBSU101 and pANIC‐10A, containing reporter genes eGFP and pporRFP, respectively. Our results revealed that the plasmid‐mediated transformation efficiencies of antibiotic‐resistant competent BCAs identified as PSL, IMC8, and PS were up 84%. Fluorescent BCA‐tagged reporter genes were associated with roots and hypocotyls but not with leaves or stems and were confirmed by fluoresence microscopy and PCR analyses in colonized Arabidopsis and sorghum. This fluorescence‐based technique's high resolution and reproducibility make it a platform‐independent system that allows tracking of BCAs spatially within plant tissues, enabling assessment of the movement and niches of BCAs within colonized plants. Steps for producing and transforming competent fluorescent BCAs, as well as the inoculation of plants with transformed BCAs, localization, and confirmation of fluorescent BCAs through fluorescence imaging and PCR, are provided in this manuscript. This study features host‐plant interactions and subsequently biological and physiological mechanisms implicated in these interactions. The maximum time to complete all the steps of this protocol is approximately 3 months.

Influence of N-terminal His-tags on the production of recombinant proteins in the cytoplasm of Bacillus subtilis

The influence of fusion tags to produce recombinant proteins in the cytoplasm of Bacillus subtilis is not well-studied as in E. coli. This study aimed to investigate the influence of His-tags with different codons on the protein production levels of the high expression gene (gfp+) and low expression gene (egfp) in the cytoplasm of B. subtilis cells. We used three different N-terminal His-tags, M-6xHis, MRGS-8xHis and MEA-8xHis, to investigate their effects on the production levels of GFP variants under the control of the Pgrac212 in B. subtilis. The fusions of His-tags with GFP+ caused a reduction compared to the construct without His-tag. When three His-tags fused with egfp, the EGFP production levels were significantly increased up to 3.5-, 12-, and 15-fold. This study suggested that His-tag at the N-terminus could enhance the protein production for the low expression gene and reduce that of the high expression gene in B. subtilis.

​​An Agrobacterium rhizogenes Strain R1000-mediated Efficient Hairy Root Transformation Protocol for Common Bean

Background: Common bean (Phaseolus vulgaris L.) is a globally important grain and vegetable legume crop, providing a substantial portion of the diet protein and minerals for many people in the developing world. However, the genetic studies and improvement on this crop has long been impeded by its recalcitrance to Agrobacterium-mediated whole plant genetic transformation. Established Agrobacterium rhizogenes-based hairy root transformation in common bean heavily relies on the strain K599. Methods: In order to develop an efficient alternative protocol for hair transformation in common bean, the efficiency of Agrobacterium rhizogenes strain R1000 in inducing hairy roots from 6-day-old seedlings with root below cotyledons excised by the soaking and smearing method were tested. The binary plasmid pBI121 with the reporter gene GUS (pBI121-GUS) or eGFP (pBI121-eGFP) driven by the constitutive promoter was used for transformation and rapid identification of the transgenic hairy roots. Result: We established a strain R1000-based system for the induction of hairy roots in common bean. The plant receptor genotypes and infection methods were optimized, which led to a high transformation rate of hairy roots up to 60%. This method therefore provides a useful alternative means for functional genomic studies in common bean.

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.

Abstract 1655: Development of humanized diffuse large B-cell lymphoma mouse models

Abstract Diffuse Large B-cell Lymphoma (DLBCL) is a commonly diagnosed aggressive non-Hodgkin’s lymphoma, with ~40% of patients experiencing the refractory or relapsed disease. The development of alternative therapies that target molecular features defining these unresponsive tumors is an active area of research to advance the field and improve clinical management significantly. However, few DLBCL animal models exist to test the efficacy of newly developed treatments and are restricted to transgenic or xenograft mice that often fail to recapitulate its sub-classifications. We explored the utility of humanizing Nod-Scid-IL2Rγnull (NSG) strains with human cytokines to establish a pipeline for the rapid, reliable generation of in vivo DLBCL models. We transduced the well-established human DLBCL cells, U2932, with the luciferase (Luc)-EGFP gene, then sorted for GFP positivity. The 1 x 106 U2932-Luc cells were injected via tail-vein into eight 12-week-old mice of various humanized NSG strains (representing equal numbers of each sex). We assessed U2932-Luc cell engraftment and growth weekly in vivo imaging (IVIS 200 Imager). To further evaluate the organ-specific engraftment/progression of U2932-Luc in the mouse, a mouse was sacrificed weekly after 15 minutes of luciferin administration. The organs were extracted and scanned for ex vivo IVIS imaging. The spleen, lung, and liver were then fixed with 10% formalin and embedded in paraffin. Sections were stained with hematoxylin, eosin, and an anti-CD20 antibody to evaluate the tumor infiltration and expansion. Unlike the previously reported DLBCL humanized strain (MISTRG6) (Hashwah et al., 2019), we included the human IL-6 expressing mouse strain. We found that both the IL6 and IL6/SGM3 strains were highly permissive to DLBCL growth. The IL-6 strain exhibited a rapid expansion of U2932 cells relative to the IL-6/SGM3 mice. The IL-6 mice survived longer than IL-6/SGM3 mice. A significant difference between the median survivals of IL-6 and IL-6/SGM3 mice (i.e., 48 vs. 42 days) was observed (p < 0.0482). The organ-specific evaluation demonstrated that U2932-Luc cells were initially engrafted and grew in the lung, liver, and spleen, subsequently found in the skeleton, ovary, and brain. Of note, we detected significant enlargements of the kidney, spleen, and ovary at the terminal stage. Our humanized mouse model approach of using U2932 human DLBCL cells transduced with the Luc gene in the NSG-IL-6 and NSG-IL-6/SGM3 mice reproduced the clinical features of an aggressive DLBCL that paralleled the original patient. This model will provide a new tool to enable the expansion of patient samples while overcoming the current limitations of DLBCL xenografts and transgenic mice. The ability to maintain the growth of patient-derived samples within clinically relevant locations has great potential to more accurately test patient-specific, personalized treatment strategies. Citation Format: Syed Hassan Mehdi, Ying-Zhi Xu, Leonard Shultz, Samantha Kendrick, Donghoon Yoon. Development of humanized diffuse large B-cell lymphoma mouse models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1655.