shRNA transgenic swine display resistance to infection with the foot-and-mouth disease virus

Foot-and-mouth disease virus (FMDV) is one of the most important animal pathogens in the world. FMDV naturally infects swine, cattle, and other cloven-hoofed animals. FMD is not adequately controlled by vaccination. An alternative strategy is to develop swine that are genetically resistant to infection. Here, we generated FMDV-specific shRNA transgenic cells targeting either nonstructural protein 2B or polymerase 3D of FMDV. The shRNA-positive transgenic cells displayed significantly lower viral production than that of the control cells after infection with FMDV (P < 0.05). Twenty-three transgenic cloned swine (TGCS) and nine non-transgenic cloned swine (Non-TGCS) were produced by somatic cell nuclear transfer (SCNT). In the FMDV challenge study, one TGCS was completely protected, no clinical signs, no viremia and no viral RNA in the tissues, no non-structural antibody response, another one TGCS swine recovered after showing clinical signs for two days, whereas all of the normal control swine (NS) and Non-TGCS developed typical clinical signs, viremia and viral RNA was determined in the tissues, the non-structural antibody was determined, and one Non-TGCS swine died. The viral RNA load in the blood and tissues of the TGCS was reduced in both challenge doses. These results indicated that the TGCS displayed resistance to the FMDV infection. Immune cells, including CD3+, CD4+, CD8+, CD21+, and CD172+ cells, and the production of IFN-γ were analyzed, there were no significant differences observed between the TGCS and NS or Non-TGCS, suggesting that the FMDV resistance may be mainly derived from the RNAi-based antiviral pathway. Our work provides a foundation for a breeding approach to preventing infectious disease in swine.

An increased proportion of transgenic plants in the progeny of rapeseed (Brassica napus L.) transformants

Cotyledon and leaf explants of two spring rapeseed varieties were transformed with Agrobacterium tumefaciens harboring a genetic construct with the gfp marker gene. In order to reduce the proportion of hyperhydrated shoots, which appeared during regenerant formation, we optimized sucrose content in the regeneration media. Analysis of the progeny obtained from T0 regenerants showed that in a number of lines the distribution of the gfp marker did not follow Mendelian segregation of a monogenic trait in self-pollinated plants, while in the progeny of the other lines of transgenic plants, the gfp marker was completely absent, although its presence had been confirmed in all selected T0 plants. We also found that in individual transformants gfp is randomly inherited throughout the central peduncle; its presence in the genome of seedlings does not depend on the location of the pod. Thus, both transformed and non-transformed cells were involved in the formation of gametes in T0 plants. In addition, marker segregation was different in plants of the T1 line obtained by nodal cuttings of a primary transformant, depending on the location of the cuttings on the stem of the original plant, indicating that the nature of T1 generation plants was also chimeric. Furthermore, we showed that propagation of plants by cutting followed by propagation by seeds formed as a result of self-pollination led to an increase in the proportion of transgenic plants in subsequent generations. The results obtained during the course of this study show that the transformants were chimeric, i. e. their tissues contained both transgenic and non-transgenic cells, and this chimeric nature was passed on to subsequent generations. We found that, in addition to nutrient media composition, other factors such as plant genotype and explant type also contribute to the rising of chimeric plants during transformation. Based on these results, we developed a simplified method, which consists of several rounds of a combination of cutting, seed production by self-pollination, and subsequent culling of wild-type plants, which significantly enriched descendent populations of the original rapeseed transformants with plants transgenic for the gfp marker.

A Synthetic Strong and Constitutive Promoter Derived from the Stellaria media pro-SmAMP1 and pro-SmAMP2 Promoters for Effective Transgene Expression in Plants

Synthetic promoters are vital for genetic engineering-based strategies for crop improvement, but effective methodologies for their creation and systematic testing are lacking. We report here on the comparative analysis of the promoters pro-SmAMP1 and pro-SmAMP2 from Stellaria media ANTIMICROBIAL PEPTIDE1 (AMP1) and ANTIMICROBIAL PEPTIDE2 (AMP2). These promoters are more effective than the well-known Cauliflower mosaic virus 35S promoter. Although these promoters share about 94% identity, the pro-SmAMP1 promoter demonstrated stronger transient expression of a reporter gene in Agrobacterium infiltration of Nicotiana benthamiana leaves, while the pro-SmAMP2 promoter was more effective for the selection of transgenic tobacco (Nicotiana tabacum) cells when driving a selectable marker. Using the cap analysis of gene expression method, we detected no differences in the structure of the transcription start sites for either promoter in transgenic plants. For both promoters, we used fine-scale deletion analysis to identify 160 bp-long sequences that retain the unique properties of each promoter. With the use of chimeric promoters and directed mutagenesis, we demonstrated that the superiority of the pro-SmAMP1 promoter for Agrobacterium-mediated infiltration is caused by the proline-inducible ACTCAT cis-element strictly positioned relative to the TATA box in the core promoter. Surprisingly, the ACTCAT cis-element not only activated but also suppressed the efficiency of the pro-SmAMP1 promoter under proline stress. The absence of the ACTCAT cis-element and CAANNNNATC motif (negative regulator) in the pro-SmAMP2 promoter provided a more constitutive gene expression profile and better selection of transgenic cells on selective medium. We created a new synthetic promoter that enjoys high effectiveness both in transient expression and in selection of transgenic cells. Intact promoters with differing properties and high degrees of sequence identity may thus be used as a basis for the creation of new synthetic promoters for precise and coordinated gene expression.

Aspartate Residues in a Forisome-Forming SEO Protein Are Critical for Protein Body Assembly and Ca2+ Responsiveness

Forisomes are protein bodies known exclusively from sieve elements of legumes. Forisomes contribute to the regulation of phloem transport due to their unique Ca2+-controlled, reversible swelling. The assembly of forisomes from sieve element occlusion (SEO) protein monomers in developing sieve elements and the mechanism(s) of Ca2+-dependent forisome contractility are poorly understood because the amino acid sequences of SEO proteins lack conventional protein–protein interaction and Ca2+-binding motifs. We selected amino acids potentially responsible for forisome-specific functions by analyzing SEO protein sequences in comparison to those of the widely distributed SEO-related (SEOR), or SEOR proteins. SEOR proteins resemble SEO proteins closely but lack any Ca2+ responsiveness. We exchanged identified candidate residues by directed mutagenesis of the Medicago truncatula SEO1 gene, expressed the mutated genes in yeast (Saccharomyces cerevisiae) and studied the structural and functional phenotypes of the forisome-like bodies that formed in the transgenic cells. We identified three aspartate residues critical for Ca2+ responsiveness and two more that were required for forisome-like bodies to assemble. The phenotypes observed further suggested that Ca2+-controlled and pH-inducible swelling effects in forisome-like bodies proceeded by different yet interacting mechanisms. Finally, we observed a previously unknown surface striation in native forisomes and in recombinant forisome-like bodies that could serve as an indicator of successful forisome assembly. To conclude, this study defines a promising path to the elucidation of the so-far elusive molecular mechanisms of forisome assembly and Ca2+-dependent contractility.

Increased metal tolerance and bioaccumulation of zinc and cadmium in Chlamydomonas reinhardtii expressing a AtHMA4 C-terminal domain protein

The use of microalgal biomass for metal pollutant bioremediation might be improved by genetic engineering to modify the selectivity or capacity of metal biosorption. A plant cadmium (Cd) and zinc (Zn) transporter (AtHMA4) was used as a transgene to increase the ability of Chlamydomonas reinhardtii to tolerate 0.2 mM Cd and 0.3 mM Zn exposure. The transgenic cells showed increased accumulation and internalisation of both metals compared to wild type. AtHMA4 was expressed either as the full-length protein or just the C-terminal tail, which is known to have metal binding sites. Similar Cd and Zn tolerance and accumulation was observed with expression of either the full-length protein or C-terminal domain, suggesting that enhanced metal tolerance was mainly due to increased metal binding rather than metal transport. The effectiveness of the transgenic cells was further examined by immobilisation in calcium alginate to generate microalgal beads that could be added to a metal contaminated solution. Immobilisation maintained metal tolerance, while AtHMA4-expressing cells in alginate showed a concentration-dependent increase in metal biosorption that was significantly greater than alginate beads composed of wild type cells. This demonstrates that expressing AtHMA4 full-length or C-terminus has great potential as a strategy for bioremediation using microalgal biomass.

135 Strategies for transfection of bovine mesenchymal stem cells with pBC1-anti-CD3 vector

Cells from different origins behave differently regarding the incorporation of exogenous genetic material and the formation of transgenic cells. In this context, the objective of this study was to verify the potential of transfection of bovine mesenchymal stem cells from Wharton's jelly and adipose tissue, comparing two transfection protocols, using Lipofectamine LTX and Plus or Xfect reagents, with the integration of humanized anti-CD3. Skin fibroblasts were used as a control group. Humanized anti-CD3 is a monoclonal antibody that interacts with the CD3 molecule of the T-cell receptor, leading to the suppression of T-cells. This antibody is considered an option in the treatment of human autoimmune diseases and against the rejection of transplanted organs. Humanized anti-CD3 was used in this work for the production of bovine transgenic cells that, in the future, will be used in the development of bioreactor animals. In all steps of this study, cell types were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS) and antibiotics, in an incubator at 39°C with 5% CO2 in air with saturated humidity. All cells were plated at 5×105 into 24-well culture dishes and co-transfected with vector pBC1-anti-CD3-IRES-FEO and pEF-NEO-GFP using Lipofectamine LTX with reagent Plus or Xfect. Forty-eight hours after transfection, neomycin was added in each treatment and cells were cultured for 2 weeks. Treated cells were submitted to fluorescence microscopy, flow cytometry, and PCR evaluations. Wharton's jelly cells were sensitive to treatments and started necrosis. In the flow cytometry assay, the median fluorescence was higher in adipocytes than in fibroblasts, for both the Xfect reagent (20.057±1.620.7 and 10.601±702.86, respectively, P&lt;0.05) and for LTX (19.590±113.84 and 10.518±442.65 respectively, P&lt;0.05). These results, associated with the evaluation of epifluorescence, demonstrated that adipocytes presented a better response to transfection than did other cells, independent of the kit used. Performing PCR on co-transfected adipocytes and fibroblasts demonstrated the presence of anti-CD3, making this approach feasible in future experiments. Southern blotting analysis is being performed to confirm DNA integration. Financial support was provided by Fundação de Amparo à Pesquisa do Distrito Federal (FAPDF); Embrapa MP1.

The Scope for Thalassemia Gene Therapy by Disruption of Aberrant Regulatory Elements

The common IVSI-110 (G>A) β-thalassemia mutation is a paradigm for intronic disease-causing mutations and their functional repair by non-homologous end joining-mediated disruption. Such mutation-specific repair by disruption of aberrant regulatory elements (DARE) is highly efficient, but to date, no systematic analysis has been performed to evaluate disease-causing mutations as therapeutic targets. Here, DARE was performed in highly characterized erythroid IVSI-110(G>A) transgenic cells and the disruption events were compared with published observations in primary CD34+ cells. DARE achieved the functional correction of β-globin expression equally through the removal of causative mutations and through the removal of context sequences, with disruption events and the restriction of indel events close to the cut site closely resembling those seen in primary cells. Correlation of DNA-, RNA-, and protein-level findings then allowed the extrapolation of findings to other mutations by in silico analyses for potential repair based on the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9, Cas12a, and transcription activator-like effector nuclease (TALEN) platforms. The high efficiency of DARE and unexpected freedom of target design render the approach potentially suitable for 14 known thalassemia mutations besides IVSI-110(G>A) and put it forward for several prominent mutations causing other inherited diseases. The application of DARE, therefore, has a wide scope for sustainable personalized advanced therapy medicinal product development for thalassemia and beyond.

The effect of BACE1-AS on β-amyloid generation by regulating BACE1 mRNA expression

Background The BACE1 antisense transcript (BACE1-AS) is a conserved long noncoding RNA (lncRNA). The level of BACE1-AS is significantly increased and the level of the BACE1 mRNA is slightly increased in subjects with AD. BACE1-AS exerts a significant moderating effect on the expression of the BACE1 mRNA and promotes the formation of Aβ. After the administration of Aβ1-42 to SH-SY5Y cells and C57/BL6J mice, we detected the expression of BACE1-AS, BACE1 mRNA, and BACE1 protein, as well as the concentration of Aβ1-40. Then, we silenced the expression of BACE1-AS in SH-SY5Y and 20E2 cells using siRNAs targeting BACE1-AS and detected its effects on the levels of the BACE1 mRNA and BACE1 protein and Aβ1-40 generation. Results The administration of Aβ1-42 increased the expression of BACE1-AS, BACE1 mRNA and protein, as well as the concentration of Aβ1-40 in SH-SY5Y cells and the brains of C57BL/6J mice. Pretreatment with the BACE1-AS siRNA inhibited the effect of Aβ1-42 on increasing the expression of BACE1-AS and BACE1, as well as the generation of Aβ. Conclusions The mechanism by which exogenous Aβ1-42 induces BACE1 expression and Aβ generation is mediated by BACE1-AS. BACE1-AS is involved in the mechanism regulating BACE1 expression and Aβ generation in APPsw transgenic cells.

A versatile bulk electrotransfection protocol for mouse embryonic fibroblast and iPS cells

A square-wave pulsing protocol was developed using OptiMEM-GlutaMAX for high efficient transfection of mouse embryonic fibroblast (MEF) and induced pluripotency stem (iPS) cells. An electrotransfection efficiency of > 95% was repeated for both MEF and iPS cells using reporter-encoding plasmids. The protocol was very efficient for plasmid size ranging from 6.2 to 13.5 kb. A high rate of targeted gene knockout (> 95 %) was produced in Venus transgenic cells using indels formation. Targeted deletions in the Venus transgene were performed by co-electroporation of two gRNA-encoding plasmids. In conclusion, this plasmid electrotransfection protocol is straight-forward, cost-effective, and efficient for CRISPRing mouse primary cells.