Transgene Integration Research Articles

CRISPR-Cas9-induced targeted mutagenesis of feruloyl CoA 6′-hydroxylase gene reduces postharvest physiological deterioration in cassava roots

Cassava is a starch-rich, multipurpose root crop grown by millions of smallholder farmers in tropical and subtropical countries. Cassava cultivation is seriously constrained by post-harvest physiological deterioration (PPD) of roots that occurs within 24–72 h after harvest, making them unpalatable and unmarketable. The inevitable wounding of roots during harvest triggers an oxidative burst throughout the root together with the accumulation of secondary metabolites with scopoletin being the most abundant. Blue-black discoloration and vascular streaking observed during PPD occur due to over-accumulation of scopoletin whose biosynthesis involves key enzymes including feruloyl CoA 6′-hydroxylase.We applied the CRISPR-Cas9 system for targeted mutagenesis of feruloyl CoA 6′-hydroxylase genes (MeF6'H1, MeF6'H12 MeF6'H2 and MeF6'H3) in cassava to delay PPD. To generate cassava plants with enhanced root shelf-life, CRISPR-Cas9 cassettes with Cas9 gene and sgRNA targeting three genes; MeF6'H1, MeF6'H2, and MeF6'H3 were introduced into cassava via Agrobacterium-mediated transformation. Integration of transgenes in putative knockout plants were confirmed by PCR while targeted sanger sequencing revealed CRISPR-Cas9 induced mutations with insertion, deletions and substitution being reported upstream and downstream of the PAM sequence of target genes. Targeted for edition of dual genes exhibited significant reduction of scopoletin below HPLC detectable levels and had improved shelf-life compared to one gene knockout events and wild-type plants. Evaluation of phenotypic variations between mutant and wild-type cassava plants revealed mutation-event associated traits including stem and petiole depigmentation, auxiliary budding, claw-like leaf appearance, loss of apical dominance and leaf chlorosis. This study demonstrated an effective and feasible approach to extend cassava root shelf-life to stabilize yield production, prevent postharvest wastage, and improve farmers’ income.

Targeted Integration of a Chimeric Antigen Receptor in Key Selected Loci in Primary Natural Killer Cells

Despite promising preclinical studies and early clinical data (Liu et al., NEJM, 2020), chimeric antigen receptor natural killer cells (CAR NK) are not yet on par with CAR T cells for the treatment of malignant diseases. Much effort has gone into developing NK-optimized CAR architectures (Li et al., Cell Stem Cell, 2018) and to increasing the persistence of CAR NK cells. We hypothesized that CAR NK cells could also benefit from the targeted integration of a CAR transgene, as compared with semi-random integration obtained using gammaretroviral vectors. Precise targeting has proven highly advantageous for TRAC CAR T cells owing to the optimal regulation and homogeneous expression conferred by the endogenous TCR alpha promoter (Eyquem et al., Nature, 2017). We first optimized an editing protocol for primary NK cells that uses electroporation of a CRISPR/Cas9 ribonucleoprotein followed by transduction with AAV6 bearing a homology-directed repair template. With this protocol we were able to knock out (KO) genes (including CD38, B2M, and different NK receptors), knock in transgenes (GFP-fusion protein, truncated EGFR, CAR), or both, at high efficiency. There was only moderate cell toxicity, and proliferation and cytotoxicity were conserved. Because of the high constitutive expression of B2M, we initially chose to focus on this locus and targeted a 1928z CAR transgene for regulation under the endogenous B2M promoter; our template design also rescues the B2M gene upon knock-in, enabling intermediate B2M expression (Figure 1). This strategy conferred high expression of the CAR and significantly more homogeneity as compared with gammaretroviral delivery (Figure 1). Furthermore, the rescued B2M expression protected B2M CAR NK cells from fratricide killing, whereas the B2M KO NK cells and the CAR NK cells with disrupted B2M expression were susceptible. B2M CAR NK cells also displayed higher in vitro cytotoxicity as compared with WT NK cells against two different CD19-expressing tumor cell lines (NALM6, SupB15). We tested B2M CAR NK cells in an NSG mouse xenograft model with an intraperitoneally injected human ovarian cancer cell line engineered to express CD19 (CD19 + SKOV3). Unexpectedly, compared to retroviral CAR-NK cells, B2M CAR NK cells conferred less tumor control (assessed by bioluminescence imaging) and less of a survival benefit. Therefore, we screened a panel of other loci for CAR targeting: five for NK activating receptors ( CD16A, CD56, DNAM1, NKp30, NKp46) and four for NK inhibitory receptors ( CD161, LILRB1, NKG2A, TIGIT). For activating receptors, the transgene was designed to use the endogenous promoter and to maintain expression of the targeted locus. For inhibitory receptors, the transgene was placed under the control of an EF1a exogenous promoter and designed to disrupt expression of the targeted locus. We compared these constructs and the initial B2M CAR NK design for CAR expression (by flow cytometry) and in vitro functionality (cytotoxicity assays) and observed wide variation in knock-in efficiency and CAR expression (Figure 2). For the top constructs based on knock-in efficiency, we conducted in vitro cytotoxicity assays against NALM6 and SupB15 cell lines. We found that when normalized for CAR percentage using the respective KO conditions, all tested knock-in loci performed better than B2M CAR NK cells, despite the lower CAR expression. Our current focus is on validating these top loci for the targeted integration of a 1928z CAR using in vivo models for comparison with B2M CAR NK and retroviral CAR NK. In summary, we have identified advantageous in vitro characteristics for the targeted integration of a CAR transgene in primary NK cells and screened 10 loci for CAR expression and in vitro cytotoxicity against tumor target cells. Confirmatory in vivo studies are ongoing and will be presented.

CRISPR-Cas12a-integrated transgenes in genomic safe harbors retain high expression in human hematopoietic iPSC-derived lineages and primary cells

Discovery of genomic safe harbor sites (SHSs) is fundamental for multiple transgene integrations, such as reporter genes, chimeric antigen receptors (CARs), and safety switches, which are required for safe cell products for regenerative cell therapies and immunotherapies. Here we identified and characterized potential SHS in human cells. Using the CRISPR-MAD7 system, we integrated transgenes at these sites in induced pluripotent stem cells (iPSCs), primary T and natural killer (NK) cells, and Jurkat cell line, and demonstrated efficient and stable expression at these loci. Subsequently, we validated the differentiation potential of engineered iPSC toward CD34+ hematopoietic stem and progenitor cells (HSPCs), lymphoid progenitor cells (LPCs), and NK cells and showed that transgene expression was perpetuated in these lineages. Finally, we demonstrated that engineered iPSC-derived NK cells retained expression of a non-virally integrated anti-CD19 CAR, suggesting that several of the investigated SHSs can be used to engineer cells for adoptive immunotherapies.

Targeted Delivery of Chimeric Antigen Receptor into T Cells via CRISPR-Mediated Homology-Directed Repair with a Dual-AAV6 Transduction System.

CAR-T cell therapy involves genetically engineering T cells to recognize and attack tumour cells by adding a chimeric antigen receptor (CAR) to their surface. In this study, we have used dual transduction with AAV serotype 6 (AAV6) to integrate an anti-CD19 CAR into human T cells at a known genomic location. The first viral vector expresses the Cas9 endonuclease and a guide RNA (gRNA) targeting the T cell receptor alpha constant locus, while the second vector carries the DNA template for homology-mediated CAR insertion. We evaluated three gRNA candidates and determined their efficiency in generating indels. The AAV6 successfully delivered the CRISPR/Cas9 machinery in vitro, and molecular analysis of the dual transduction showed the integration of the CAR transgene into the desired location. In contrast to the random integration methods typically used to generate CAR-T cells, targeted integration into a known genomic locus can potentially lower the risk of insertional mutagenesis and provide more stable levels of CAR expression. Critically, this method also results in the knockout of the endogenous T cell receptor, allowing target cells to be derived from allogeneic donors. This raises the exciting possibility of "off-the-shelf" universal immunotherapies that would greatly simplify the production and administration of CAR-T cells.

Agrobacterium-Mediated Transformation for the Development of Transgenic Crops; Present and Future Prospects.

Plant transformation based on Agrobacterium-mediated transformation is a technique that mimics the natural agrobacterium system for gene(s) introduction into crops. Through this technique, various crop species have been improved/modified for different trait/s, showing a successful genetic transformation so far. This technique has many advantages over other transformation methods such as stable integration of transgene, cost effective. However, there are many limitations of this technology such as mostly the crops are recalcitrant to agrobacterium, low transformation efficiency, transgene integration as well as off targets. So, it's very important to explore the major limitations and possible solutions for Agrobacterium-mediated transformation in order to increase its genetic transformation efficiency. Therefore, the present review article gives a comprehensive study how the transgenic crops are developed using Agrobacterium-mediated transformation, crops that have already been modified through this method, and risks associated with transgenic plants based on Agrobacterium-mediated transformation. Moreover, the challenges and problems associated with Agrobacterium-mediated transformation and how those problems can be solved in future for a successful genetic transformation of crops using modern biotechnology techniques such as CRISPR/Cas9 systems. The present review article will be really helpful for the audience those working on Genome editing of crops using Agrobacterium-mediated transformation and will opens many ways for future plant genetic transformation.

High-efficiency transgene integration by homology-directed repair in human primary cells using DNA-PKcs inhibition.

Therapeutic applications of nuclease-based genome editing would benefit from improved methods for transgene integration via homology-directed repair (HDR). To improve HDR efficiency, we screened six small-molecule inhibitors of DNA-dependent protein kinase catalytic subunit (DNA-PKcs), a key protein in the alternative repair pathway of non-homologous end joining (NHEJ), which generates genomic insertions/deletions (INDELs). From this screen, we identified AZD7648 as the most potent compound. The use of AZD7648 significantly increased HDR (up to 50-fold) and concomitantly decreased INDELs across different genomic loci in various therapeutically relevant primary human cell types. In all cases, the ratio of HDR to INDELs markedly increased, and, in certain situations, INDEL-free high-frequency (>50%) targeted integration was achieved. This approach has the potential to improve the therapeutic efficacy of cell-based therapies and broaden the use of targeted integration as a research tool.

Characterization of integration sites and transfer DNA structures in Agrobacterium-mediated transgenic events of maize inbred B104.

In maize, the community-standard transformant line B104 is a useful model for dissecting features of transfer DNA (T-DNA) integration due to its compatibility with Agrobacterium-mediated transformation and the availability of its genome sequence. Knowledge of transgene integration sites permits the analysis of the genomic environment that governs the strength of gene expression and phenotypic effects due to the disruption of an endogenous gene or regulatory element. In this study, we optimized a fusion primer and nested integrated PCR (FPNI-PCR) technique for T-DNA detection in maize to characterize the integration sites of 89 T-DNA insertions in 81 transformant lines. T-DNA insertions preferentially occurred in gene-rich regions and regions distant from centromeres. Integration junctions with and without microhomologous sequences as well as junctions with de novo sequences were detected. Sequence analysis of integration junctions indicated that T-DNA was incorporated via the error-prone repair pathways of nonhomologous (predominantly) and microhomology-mediated (minor) end-joining. This report provides a quantitative assessment of Agrobacterium-mediated T-DNA integration in maize with respect to insertion site features, the genomic distribution of T-DNA incorporation, and the mechanisms of integration. It also demonstrates the utility of the FPNI-PCR technique, which can be adapted to any species of interest.

Strategies for delivery of CRISPR/Cas-mediated genome editing to obtain edited plants directly without transgene integration.

Increased understanding of plant genetics and the development of powerful and easier-to-use gene editing tools over the past century have revolutionized humankind's ability to deliver precise genotypes in crops. Plant transformation techniques are well developed for making transgenic varieties in certain crops and model organisms, yet reagent delivery and plant regeneration remain key bottlenecks to applying the technology of gene editing to most crops. Typical plant transformation protocols to produce transgenic, genetically modified (GM) varieties rely on transgenes, chemical selection, and tissue culture. Typical protocols to make gene edited (GE) varieties also use transgenes, even though these may be undesirable in the final crop product. In some crops, the transgenes are routinely segregated away during meiosis by performing crosses, and thus only a minor concern. In other crops, particularly those propagated vegetatively, complex hybrids, or crops with long generation times, such crosses are impractical or impossible. This review highlights diverse strategies to deliver CRISPR/Cas gene editing reagents to regenerable plant cells and to recover edited plants without unwanted integration of transgenes. Some examples include delivering DNA-free gene editing reagents such as ribonucleoproteins or mRNA, relying on reagent expression from non-integrated DNA, using novel delivery mechanisms such as viruses or nanoparticles, using unconventional selection methods to avoid integration of transgenes, and/or avoiding tissue culture altogether. These methods are advancing rapidly and already enabling crop scientists to make use of the precision of CRISPR gene editing tools.

CRISPR/Cas9-mediated knock-in of masu salmon (Oncorhyncus masou) elongase gene in the melanocortin-4 (mc4r) coding region of channel catfish (Ictalurus punctatus) genome.

Channel catfish, Ictalurus punctatus, have limited ability to synthesize Ω-3 fatty acids. The ccβA-msElovl2 transgene containing masu salmon, Oncorhynchus masou, elongase gene driven by the common carp, Cyprinus carpio, β-actin promoter was inserted into the channel catfish melanocortin-4 receptor (mc4r) gene site using the two-hit two-oligo with plasmid (2H2OP) method. The best performing sgRNA resulted in a knockout mutation rate of 92%, a knock-in rate of 54% and a simultaneous knockout/knock-in rate of 49%. Fish containing both the ccβA-msElovl2 transgene knock-in and mc4r knockout (Elovl2) were 41.8% larger than controls at 6months post-hatch (p = 0.005). Mean eicosapentaenoic acid (EPA, C20:5n-3) levels in Elov2 mutants and mc4r knockout mutants (MC4R) were 121.6% and 94.1% higher than in controls, respectively (p = 0.045; p = 0.025). Observed mean docosahexaenoic acid (DHA, C22:6n-3) and total EPA + DHA content was 32.8% and 45.1% higher, respectively, in Elovl2 transgenic channel catfish than controls (p = 0.368; p = 0.025). To our knowledge this is the first example of genome engineering to simultaneously target transgenesis and knock-out a gene in a commercially important aquaculture species for multiple improved performance traits. With a high transgene integration rate, improved growth, and higher omega-3 fatty acid content, the use of Elovl2 transgenic channel catfish appears beneficial for application on commercial farms.

High-efficiency targeted transgene integration via primed micro-homologues

Due to the difficulties in precisely manipulating DNA repair pathways, high-fidelity targeted integration of large transgenes triggered by double-strand breaks is inherently inefficient. Here, we exploit prime editors to devise a robust knock-in (KI) strategy named primed micro-homologues-assisted integration (PAINT), which utilizes reverse-transcribed single-stranded micro-homologues to boost targeted KIs in different types of cells. The improved version of PAINT, designated PAINT 3.0, maximizes editing efficiency and minimizes off-target integration, especially in dealing with scarless in-frame KIs. Using PAINT 3.0, we target a reporter transgene into housekeeping genes with editing efficiencies up to 80%, more than 10-fold higher than the traditional homology-directed repair method. Moreover, the use of PAINT 3.0 to insert a 2.5-kb transgene achieves up to 85% KI frequency at several therapeutically relevant genomic loci, suggesting its potential for clinical applications. Finally, PAINT 3.0 enables high-efficiency non-viral genome targeting in primary T cells and produces functional CAR-T cells with specific tumor-killing ability. Thus, we establish that the PAINT method is a powerful gene editing tool for large transgene integrations and may open new avenues for cell and gene therapies and genome writing technologies.

Integration of alligator cathelicidin gene via two CRISPR/Cas9-assisted systems enhances bacterial resistance in blue catfish, Ictalurus furcatus

CRISPR/Cas9-mediated genome editing has paved new avenues for improving production-valued traits in aquaculture by knocking out or disrupting functional genes. However, utilizing CRISPR/Cas9-based knock-in (KI) of exogenous genes can expedite genetic improvement of traits such as disease resistance, which remains problematic in farmed fish. In this study, we successfully generated transgenic blue catfish (Ictalurus furcatus) of primarily Rio Grande strain ancestry with site-specific KI of the alligator (Alligator sinensis) cathelicidin (As-Cath) gene into the luteinizing hormone (lh) locus via two CRISPR/Cas9-mediated KI systems, assisted by the linear double-stranded DNA (dsDNA) and double-cut plasmid, respectively. High integration rates were observed with linear dsDNA (16.67%, [13/78]) and double-cut plasmid strategies (24.53%, [26/106]). In addition, the on-target KI efficiency of the double-cut plasmid strategy (16.04%, [17/106]) was 1.67 times higher than that of the linear dsDNA strategy (10.26%, [8/78]) based on the odds ratio. The relative expression of the As-Cath transgene of P1 founders was detected in nine tissues, dominated by the kidney, skin, and muscle (14.30-, 7.71- and 6.92-fold change, P < 0.05). Moreover, the As-Cath transgenic blue catfish showed a higher cumulative survival rate than that of wild-type controls (80% vs. 30%, P < 0.05) following Flavobacterium covae infection. Survival during culture supports the challenge data as survival of As-Cath transgenic individuals was 97.1% while that of pooled non-transgenic individuals was observed to be less 87.0% (P = 0.15). The growth rates and external morphology of the transgenic and wild-type siblings were not different (P > 0.05), indicating no pleiotropic effects of the As-Cath transgene integration at the lh locus in the P1 founders for this trait. Taken together, our findings demonstrate that CRISPR/Cas9-assisted KI of an antimicrobial peptide gene can be achieved in blue catfish with high integration efficiency, and As-Cath transgenic blue catfish have improved disease resistance, which is a promising strategy for disease reduction in aquaculture.

A splinkerette PCR-based genome walking technique for the identification of transgene integration sites in CHO cells.

Identification of recombinant gene integrations sites in the Chinese hamster ovary (CHO) cell genome is increasingly important to assure monoclonality. While next-generation sequencing (NGS) is commonly used for the gene integration site analysis, it is a time-consuming and costly technique as it analyzes the entire genome. Hence, simple, easy, and inexpensive methods to analyze transgene insertion sites are necessary. To selectively capture the integration site of transgene in the CHO genome, we applied splinkerette-PCR (spPCR). SpPCR is an adaptor ligation-based method using splinkerette adaptors that have a stable hairpin loop. Restriction enzymes with high frequencies in the CHO genome were chosen using a Python script and used for the in vitro spPCR assay development. After testing on two CHO housekeeping genes with known loci, the spPCR-based genome walking technique was successfully applied to recombinant CHO cells to identify the transgene integration site. Finally, the comparison with NGS methods exhibited that the time and cost required for the analysis can be substantially reduced. Taken together, the established technique would aid the stable cell line development process by providing a rapid and cost-effective method for transgene integration site analysis.

Engineered ketocarotenoid biosynthesis in the polyextremophilic red microalga Cyanidioschyzon merolae 10D

The polyextremophilic Cyanidiophyceae are eukaryotic red microalgae with promising biotechnological properties arising from their low pH and elevated temperature requirements which can minimize culture contamination at scale. Cyanidioschyzon merolae 10D is a cell wall deficient species with a fully sequenced genome that is amenable to nuclear transgene integration by targeted homologous recombination. C. merolae maintains a minimal carotenoid profile and here, we sought to determine its capacity for ketocarotenoid accumulation mediated by heterologous expression of a green algal β-carotene ketolase (BKT) and hydroxylase (CHYB). To achieve this, a synthetic transgene expression cassette system was built to integrate and express Chlamydomonas reinhardtii (Cr) sourced enzymes by fusing native C. merolae transcription, translation and chloroplast targeting signals to codon-optimized coding sequences. Chloramphenicol resistance was used to select for the integration of synthetic linear DNAs into a neutral site within the host genome. CrBKT expression caused accumulation of canthaxanthin and adonirubin as major carotenoids while co-expression of CrBKT with CrCHYB generated astaxanthin as the major carotenoid in C. merolae. Unlike green algae and plants, ketocarotenoid accumulation in C. merolae did not reduce total carotenoid contents, but chlorophyll a reduction was observed. Light intensity affected global ratios of all pigments but not individual pigment compositions and phycocyanin contents were not markedly different between parental strain and transformants. Continuous illumination was found to encourage biomass accumulation and all strains could be cultivated in simulated summer conditions from two different extreme desert environments. Our findings present the first example of carotenoid metabolic engineering in a red eukaryotic microalga and open the possibility for use of C. merolae 10D for simultaneous production of phycocyanin and ketocarotenoid pigments.

Efficient Agrobacterium mediated genetic transformation of CRISPR/Cas9 construct using cotyledonary node explants of greengram

Efficient Agrobacterium mediated genetic transformation of CRISPR/Cas9 construct using cotyledonary node explants of greengram

903-P: Correlation of Gene Expression and Cellular Pathways in Insulin-Expressing Mouse Liver Cells Transduced with an Integrating Adeno-associated Viral Vector (AAV)

Gene therapy is being considered as a treatment/cure for type 1 diabetes (T1D). Previously we used a clinically-applicable AAV to deliver furin cleavable insulin (INS-FUR) to the livers of diabetic non-obese diabetic mice. The traditional AAV8 that allows predominantly episomal expression, and the hybrid AAV8/piggyBac that results in transgene integration into the host genome were used. Euglycaemia was achieved in mice (n=4) that received AAV8/piggyBac-INS-FUR, but not in mice that received AAV8-INS-FUR. In the current study global transcriptomic profiling was carried out, by total RNA sequencing, to compare the key pathways modulated in hepatocytes that reversed diabetes with those that did not. Using Qiagen CLC Workbench, significant genes were identified based on fold change expression cut offs of, and a false discovery rate p-value of &amp;lt;0.05. Selected genes were mapped onto the various cellular pathways using Qiagen Ingenuity Pathway Analysis (IPA) software to determine trends in pathway activation/ inactivation related to the altered gene expression. IPA analysis showed that the canonical pathways identified between the treatments remained largely the same, however their downstream biological processes differed. Key canonical pathways identified include pathways related to insulin signalling, glucose and lipid metabolism. Key pathway players identified as significantly altered in the comparative treatments included insulin, major histocompatibility complex class I, A, glucose-6-phosphatase catalytic subunit 1 and insulin like growth factor binding protein 1. The profile of AAV8/piggyBac-INS-FUR transduced mouse livers resembled that of a normal liver compared to livers transduced with AAV8-INS-FUR. This study confirms the correlation of transcriptomic data mapping and has identified differences in the cellular effects of both vector systems used to deliver INS-FUR for T1D treatment. Disclosure S.Joshua: None. N.T.Nassif: None. A.M.Simpson: None. Funding National Health and Medical Research Council of Australia (1086256)

1684-P: Transgene Integration-Induced Genomic Rearrangement in a Noncoding Region of Chromosome 1 Leads to Spontaneous Obesity and Type 2 Diabetes in Mice

Obesity is a critical risk factor for type 2 diabetes (T2D) and has become a global health issue. We found that a mouse line (C57/B6J) of Floxed-stop over expression of IF1 (ATPase Inhibitory Factor 1) (hereon called FEI) develops spontaneous obesity without induction of transgenic expression. Targeted-Locus-Amplification (TLA) analyses revealed a rearrangement in upstream sequences of the transgene integration site, and a duplication downstream in a non-coding region of chromosome 1. Male and female FEI mice show early dyslipidemia before weight gain at two months, reaching 50-60 grams at 6-8 months. Body composition revealed FEI mice have markedly increased fat mass and modestly increased lean mass, consistent with morphometric findings of substantially increased peripheral fat pads. FEI mice were hyperphagic. When subjected to pair-feeding, they reverted to comparable WT body weight levels, then gained weight again when fed ad libidum. Adult FEI mice maintain comparable physical activity levels to their WT counterparts, but indirect metabolic levels among FEI mice declined. These mice exhibited glucose and insulin tolerance at six weeks of age, as well as hyperglycemia, hyper-insulinemia, and hyper-leptinemia. Histology H&amp;E staining revealed markedly enlarged adipocytes with crown-like structures on visceral white adipose tissue sections, and increased fat deposition in liver sections. RNAseq analyses from subcutaneous white adipose tissues of 5-month-old FEI mice demonstrate upregulation of adipogenic and inflammatory genes. Isolated FEI preadipocytes exhibited increased proliferation but impaired lipid deposition. RT-PCR revealed upregulation of inflammatory genes (IFI202b, TNF-α, and IL6). Together, we show FEI mice are a novel obese mouse model. Preclinical studies using this model may help yield insights into novel pathogenic mechanisms and effective treatments for obesity. Disclosure D.White: None. S.Alahari: None. E.D.Lazartigues: None. Q.Yang: None. J.Wu: None. S.Kang: None. L.Lauterboeck: None. P.Mobasheran: None. F.Paliarin: None. T.Nguyen: None. M.Elgazzaz: None. R.Maiya: None. Funding American Diabetes Association (1-17-IBS-184 to Q.Y.)

Optimization of piggyBac transposon-mediated gene transfer method in common marmoset embryos.

Generating non-human primate models of human diseases is important for the development of therapeutic strategies especially for neurodegenerative diseases. The common marmoset has attracted attention as a new experimental animal model, and many transgenic marmosets have been produced using lentiviral vector-mediated transgenesis. However, lentiviral vectors have a size limitation of up to 8 kb in length for transgene applications. Therefore, the present study aimed to optimize a piggyBac transposon-mediated gene transfer method in which transgenes longer than 8 kb were injected into the perivitelline space of marmoset embryos, followed by electroporation. We constructed a long piggyBac vector carrying the gene responsible for Alzheimer's disease. The optimal weight ratio of the piggyBac transgene vector to the piggyBac transposase mRNA was examined using mouse embryos. Transgene integration into the genome was confirmed in 70.7% of embryonic stem cells established from embryos injected with 1000 ng of transgene and transposase mRNA. Under these conditions, long transgenes were introduced into marmoset embryos. All embryos survived after transgene introduction treatment, and transgenes were detected in 70% of marmoset embryos. The transposon-mediated gene transfer method developed in this study can be applied to the genetic modification of non-human primates, as well as large animals.

Co-Culture and Transduction of Murine Thymocytes on Delta-Like 4-Expressing Stromal Cells to Study Oncogenes in T-Cell Leukemia.

Transduced mouse immature thymocytes can be differentiated into T cellsin vitrousing the delta-like 4-expressing bone marrow stromal cell line co-culture system (OP9-DL4). As retroviral transduction requires dividing cells for transgene integration, OP9-DL4 provides a suitable in vitro environment for cultivating hematopoietic progenitor cells. This is particularly advantageous when studying the effects of the expression of a specific gene during normal T cell development and leukemogenesis, as it allows researchers to circumvent the time-consuming process of generating transgenic mice. To achieve successful outcomes, a series of coordinated steps involving the simultaneous manipulation of different types of cells must be carefully performed. Although these are very well-established procedures, the lack of a common source in the literature often means a series of optimizations are required, which can be time-consuming. This protocol has been shown to be efficient in transducing primary thymocytes followed by differentiation on OP9-DL4 cells. Detailed here is a protocol that can serve as a quick and optimized guide for the co-culture of retrovirally transduced thymocytes on OP9-DL4 stromal cells.

Exploring the Potential of Plant-Based CTB-INS Oral Vaccines in Treating Type 1 Diabetes

The 19th century saw the development of vaccines, which were biological preparations designed to enhance immunity against specific diseases. Edible vaccines function by stimulating both systemic and mucosal immune responses against foreign pathogens, and they may potentially protect the host from autoimmunity. The mucosal surfaces provide a convenient and rapid route for delivering therapeutic small molecules. This is due to their large surface areas and easy administration. The effectiveness of mucosal immunization relies on the fact that mucous membranes represent the body’s largest immunogenic organ. Within this interface, there is a well-organized lymphatic structure known as MALT (mucosa-associated lymphoid tissue), which includes both T and B cells and encompasses the adaptive arms of the immune system. Oral vaccines specifically stimulate immune responses in the gut-associated lymphoid tissue (GALT), which consists of lymph nodes, Payer’s patches (where B cells make up about 75% of the population and T cells account for approximately 20%), and isolated lymphoid follicles within the gastrointestinal tract (GIT). However, a significant challenge in developing vaccines is the rapid degradation of antigens within the harsh environment of the digestive tract, which hampers effective protein delivery to the GIT. In light of recent proteomic analysis revealing strong up-regulation of the tryptophan catabolic enzyme indoleamine 2, 3-dioxygenase (IDO1) in DCs inoculated with the Cholera toxin B-subunit-Insulin fusion protein vaccine (CTB-INS), we are interested in investigating the effects of transgene integration into a selected plant cell as an edible vaccine.

Plastid engineering using episomal DNA.

Novel episomal systems have the potential to accelerate plastid genetic engineering for application in plant synthetic biology. Plastids represent valuable subcellular compartments for genetic engineering of plants with intrinsic advantages to engineering the nucleus. The ability to perform site-specific transgene integration by homologous recombination (HR), coordination of transgene expression in operons, and high production of heterologous proteins, all make plastids an attractive target for synthetic biology. Typically, plastid engineering is performed by homologous recombination; however, episomal-replicating vectors have the potential to accelerate the design/build/test cycles for plastid engineering. By accelerating the timeline from design to validation, it will be possible to generate translational breakthroughs in fields ranging from agriculture to biopharmaceuticals. Episomal-based plastid engineering will allow precise single step metabolic engineering in plants enabling the installation of complex synthetic circuits with the ambitious goal of reaching similar efficiency and flexibility of to the state-of-the-art genetic engineering of prokaryotic systems. The prospect to design novel episomal systems for production of transplastomic marker-free plants will also improve biosafety for eventual release in agriculture.