Expression of Phytase gene in transgenic maize with seed-specific promoter 27-kDa γ Zein and constitutive promoter CaMV 35S

Phytase enzymes are applied to animal feed to help animals absorb more nutrients. The use of feed raw materials containing phytase enzymes is expected to reduce the cost of animal feed production. Efforts to increase the phytase content in maize were carried out by improving genetics, in the way of assembling transgenic plants containing high phytase content. The 27-kDa γ Zein promoter is a specific promoter that expresses genes in caryopsis, and promoter CaMV 35S is a constitutive promoter that controls gene expression in all tissues and generally does not depend on the growth phase. Transgenic maize was transformed using Agrobacterium tumefacien infection method on maize B104. The reverse transcriptase polymerase chain reaction (RT-PCR) approach was used to examine the expression of phytase genes in leaves, roots, and caryopsis was done 10, 20, and 30 days after pollination (DAP). The phytase enzyme activity test was also carried out by using the colorimetric phosphomolybdate analysis method to see the phytase enzyme activity in unit µg-1. The results showed that the phytase gene in transgenic plants with the 27-kDa γ Zein promoter was highly expressed in maize caryopsis, but in line Z6.10 was also expressed in leaves, while in the CaMV 35S promoter the phytase gene was only expressed on the leaves. Phytase enzyme activity showed that transgenic maize was higher than non-transgenic maize.

Identification and Evaluation of a Panel of Strong Constitutive Promoters in Listeria monocytogenes for Improving the Expression of Foreign Antigens

Attenuated Listeria monocytogenes (L. monocytogenes) could be used as a vaccine vector for immunotherapy of tumors or pathogens. However, the lack of reliable promoters limits its ability to express foreign antigens. In this work, 21 promoters from L. monocytogenes were identified by RNA-seq analysis under two conditions of pH 7.4 and pH 5.5. Based on the constructed fluorescence report system, 7 constitutive promoters showed higher strength than that of Phelp, a previously reported strong promoter. Further, the selected 5 constitutive promoters also showed high activity in the production of UreB, a widely used antigen against Helicobacter pylori (H. pylori). In particular, a well-characterized constitutive promoter P18, which performed best in both fluorescence intensity and UreB production, was proved to be highly active in vitro and in vivo. In summary, we provide a useful promoter library for Listeria species and offer a reference for constitutive promoter mining in other organisms.Key points21 promoters from L. monocytogenes were identified by RNA-seq.Fluorescent tracer of L. monocytogenes (P18) was performed in vitro and in vivo.A well-characterized constitutive promoter P18 could improve the expression level of a foreign antigen UreB in L. monocytogenes

How to drive phloem gene expression? A case study with preferentially expressed citrus gene promoters

New approaches for developing disease-resistant genetically modified organisms have included specific targets for gene expression to enhance the chances for pathogen control. Gene expression driven by phloem-derived Citrus sinensis gene promoters could be evaluated and compared with the expression induced by a strong constitutive promoter in the same tissue, leading to the production of transgenic sweet oranges potentially more resistant to diseases caused by phloem-limited bacteria. ‘Carrizo’ citrange [ (Poncirus trifoliataL.) Raf. x Citrus sinensis (L.) Osbeck] was transformed, via Agrobacterium tumefaciens, with the binary vector pCAMBIA2301 bearing the uidA gene (ß-glucuronidase) driven by the CaMV35S constitutive promoter (CaMV35S::uidA) or by the CsPP2.B1 (CsPP2.B1::uidA) or by the CsVTE2 (CsVTE2::uidA) citrus promoters. In vitro regenerated shoots were grafted onto ‘Rangpur’ lime (C. limonia Osbeck). The genetic transformation was confirmed by Southern blot analyses. uidA gene expression was evaluated by RT-qPCR, and gene histolocalization controlled by these three promoters was accessed by X-GLUC treated stem sections. uidA gene expression exhibited by tissue-specific promoters was overall lower than from the constitutive promoter CaMV35; however, constructs driven by tissue-specific promoters may lead to expression in restricted tissues. CsPP2.B1 and CsVTE2 promoters can be considered adequate for the utilization in gene constructs aiming disease resistance.

O6 Expression of anti-apoptotic gene cFLIP to enhance persistence in CAR T cells

BackgroundCAR T cell therapy has been successful for targeting blood cancers, but treatment of solid cancers has been limited due to the heterogenous nature of tumour-associated antigen expression on solid cancers, and the suppressive tumour microenvironment.1 Another major obstacle to CAR T cell therapy is activation-induced cell death (AICD) of the CAR T cells.2 In this study, we expressed the anti-apoptotic cellular FLICE-like inhibitory protein (c-FLIP short; c-FLIPs) together with the CAR construct to enhance CAR T cell persistence.3Materials and MethodsThe anti-Her2 FRP5 CAR T construct with P2A-linked cFLIPs or cFLIPp43 was cloned into the Sleeping Beauty (SB) transposon vector (pSBtet-GP) or lentiviral vector, under the control of either a tet-on or a constitutive promoter. Construct expression was validated by qPCR and immunoblot analysis. CAR T cells were generated by SB transposition or lentiviral transduction of CD3/CD28 stimulated primary human T cells that were subsequently maintained with IL-2. Mitochondrial function and apoptosis were determined by resazurin assay and by flow cytometry using tetramethyl rhodamine (TMRE).ResultsOverexpression of cFLIP (cFLIPp43 and cFLIPs) in pSBtet-GP demonstrated protection in both Jurkat T cell line and primary human T cells. pSBtet-GP was modified to overexpress cFLIPs and cFLIPp43 under tet-on promoter, with the anti-her2 CAR, GFP and rtTA under constitutive promoter. Transfer of the inducible cassette from the SB transposon to a lentiviral system resulted in a significant loss of tightness. Doxycycline treated CAR T cells showed only ~13-fold overexpression of cFLIPs or cFLIPp43 compared to untreated cells, and doxycycline significantly inhibited (approximately 30%) primary CAR T cell expansion. In contrast, constitutive expression of CAR-cFLIPs or cFLIPp43 construct gave a >3 × 105-fold cFLIP overexpression, as compared to CAR-only control. While the transduction efficiency of CAR-only was around 70–80% control in primary T cells, this dropped to 20–25% when using the more genetically complex tet-on system.ConclusionscFLIP protects T cells from Fas-induced apoptosis. The tet-on system demonstrates several drawbacks in the lentiviral system, including toxicity of the inducer drug (and/or squelching effects resulting in lowered viability), loss of responsiveness and lowered transduction frequencies. Therefore, a constitutive promoter system is preferred in lentiviral systems for the control of genes of interest within CAR T cells, while the SB transposon system may be preferred for tet-on control within CAR T cells.ReferencesPitt JM, Marabelle A, Eggermont A, Soria JC, Kroemer G, and Zitvogel L. Targeting the tumor microenvironment: removing obstruction to anticancer immune responses and immunotherapy. Ann Oncol 2016;27:1482–1492.Yeku O, Li X, Brentjens RJ. Adoptive T-Cell Therapy for Solid Tumors. Am Soc Clin Oncol Educ Book 2017;37:193–204.Dohrman A, Kataoka T, Cuenin S, Russell JQ, Tschopp J, and Budd RC. Cellular FLIP (long form) regulates CD8+ T cell activation through caspase-8-dependent NF-kappa B activation. J Immunol 2005; 174:5270–5278.Disclosure InformationG.M.Y. Tan: None. S.M.A.R. Hosseini: None. A. Poudel: None. A.D. Mclellan: None.

Effects of Mini-Dystrophin on Dystrophin-Deficient, Human Skeletal Muscle-Derived Cells

Background: We are developing a novel therapy for Duchenne muscular dystrophy (DMD), involving the transplantation of autologous, skeletal muscle-derived stem cells that have been genetically corrected to express dystrophin. Dystrophin is normally expressed in activated satellite cells and in differentiated muscle fibres. However, in past preclinical validation studies, dystrophin transgenes have generally been driven by constitutive promoters that would be active at every stage of the myogenic differentiation process, including in proliferating muscle stem cells. It is not known whether artificial dystrophin expression would affect the properties of these cells. Aims: Our aims are to determine if mini-dystrophin expression affects the proliferation or myogenic differentiation of DMD skeletal muscle-derived cells. Methods: Skeletal muscle-derived cells from a DMD patient were transduced with lentivirus coding for mini-dystrophins (R3–R13 spectrin-like repeats (ΔR3R13) or hinge2 to spectrin-like repeats R23 (ΔH2R23)) with EGFP (enhanced green fluorescence protein) fused to the C-terminus, driven by a constitutive promoter, spleen focus-forming virus (SFFV). Transduced cells were purified on the basis of GFP expression. Their proliferation and myogenic differentiation were quantified by ethynyl deoxyuridine (EdU) incorporation and fusion index. Furthermore, dystrophin small interfering ribonucleic acids (siRNAs) were transfected to the cells to reverse the effects of the mini-dystrophin. Finally, a phospho-mitogen-activated protein kinase (MAPK) array assay was performed to investigate signalling pathway changes caused by dystrophin expression. Results: Cell proliferation was not affected in cells transduced with ΔR3R13, but was significantly increased in cells transduced with ΔH2R23. The fusion index of myotubes derived from both ΔR3R13- and ΔH2R23 -expressing cells was significantly compromised in comparison to myotubes derived from non-transduced cells. Dystrophin siRNA transfection restored the differentiation of ΔH2R23-expressing cells. The Erk1/2- signalling pathway is altered in cells transduced with mini-dystrophin constructs. Conclusions: Ectopic expression of dystrophin in cultured human skeletal muscle-derived cells may affect their proliferation and differentiation capacity. Caution should be taken when considering genetic correction of autologous stem cells to express dystrophin driven by a constitutive promoter.

Construction and Cloning of Plastic-degrading Recombinant Enzymes (MHETase)

Background: Polyethylene terephthalate (PET) is the most widely produced polyester plastic in the world. PET is very difficult to catalyze or biological depolymerization due to the limited access to ester bonds. Consequently, plastic will be stockpiled or flowed into the environment which is projected until hundreds of years. The most effective and environmental friendly plastic degradation method is biodegradation with microorganisms. Two specific enzyme for PET hydrolase, PETase and MHETase have been identified from Ideonella sakaiensis 201-F6. Recombinant genes are made to increase the effectiveness of enzymes in degrading PET. Previous studies of the PETase gene have been carried out, but to produce the final degradation PET product, the enzyme MHETase is needed. Thus, in this study the MHETase gene construction was carried out. Methods: The goal of this study is to construct MHETase gene in pUCIDT plasmid with native signal peptide from I. sakaensis 201-F6 and constitutive promoter J23106 was expressed in Escherichia coli BL21 (DE3) by heats shock. Expression analysis using SDS-PAGE and activity of enzyme is analyzed by spectrophotometry method and SEM. Results: MHETase gene protein was successfully constructed in pUCIDT +Amp plasmid with native signal peptide from Ideonella sakaensis 201-F6, T7 terminator and constitutive promoter J23106. PCR analysis showed that the gene successfully contained in the cells by band size (1813 bp) in electrophoresis gel. Analysis using Snap Gene, pairwise alignment using MEGA X, and NCBI was demonstrated that MHETase sequence the gene was in-frame in pUCIDT plasmid. Conclusion: MHETase gene was successfully constructed in plasmids by in silico method. Synthetic plasmids transformed in E. coli BL21 (DE3) contain MHETase gene sequences which were in frame. Hence, the E. coli BL21 (DE3) cells have the potential to produce MHETase proteins for the plastic degradation testing process. We will patent the construct of MHETase gene using constitutive promoter and signal peptide from native which expressed in E. coli BL21 (DE3). This patent refers to a more applicable plastic degradation system with a whole cell without the need for purification and environmental conditioning of pure enzymes.