Genetically engineered E. coli invade epithelial cells and transfer their genetic cargo into the cells: an approach to a gene delivery system.

PURPOSE: Despite advances in gene therapy, the lack of safe and efficient gene delivery systems limited the clinical effectiveness of gene therapy. Due to the inherent potential of bacteria, they can be considered as a good option for the gene transfer system. This study aimed to create a genetically engineered bacterium capable of entering epithelial cells and transferring its genetic cargo to the cell's cytoplasm, eventually expressing the gene of interest in the cell. METHODS: The invasin (inv) gene from Yersinia pseudotuberculosis and the listeriolysin (hlyA) gene from Listeria monocytogenes were isolated by PCR assay and inserted into a pACYCDuet-1 vector. The recombinant plasmid was then transformed into E. coli strain BL21. Subsequently, pEGFP-C1 plasmids containing a CMV promoter were transformed into the engineered bacteria. Finally, the engineered bacteria containing the reporter genes were incubated with the HeLa and LNCaP cell lines. Fluorescence microscopy, flow cytometry, and TEM were used to monitor bacterial entry into the cells and gene expression. We used native E. coli strain BL21 as a control. RESULTS: A fluorescence microscope showed that, in contrast to the control group, the manipulated E. coli were able to penetrate the cells and transport the plasmid pEGFP-C1 to the target cells. Flow cytometry also showed fluorescence intensity of 54.7% in HeLa cells and 71% in LNCaP cells, respectively. In addition, electron micrographs revealed the presence of bacteria in the cell endosomes and in the cytoplasm of the cells. CONCLUSION: This study shows that genetically engineered E. coli can enter cells, transport cargo into cells, and induce gene expression in the target cell. In addition, flow cytometry shows that the gene transfer efficiency was sufficient for protein expression.

CRISPR System: A High-throughput Toolbox for Research and Treatment of Parkinson's Disease.

In recent years, the innovation of gene-editing tools such as the CRISPR/Cas9 system improves the translational gap of treatments mediated by gene therapy. The privileges of CRISPR/Cas9 such as working in living cells and organs candidate this technology for using in research and treatment of the central nervous system (CNS) disorders. Parkinson's disease (PD) is a common, debilitating, neurodegenerative disorder which occurs due to loss of dopaminergic neurons and is associated with progressive motor dysfunction. Knowledge about the pathophysiological basis of PD has altered the classification system of PD, which manifests in familial and sporadic forms. The first genetic linkage studies in PD demonstrated the involvement of Synuclein alpha (SNCA) mutations and SNCA genomic duplications in the pathogenesis of PD familial forms. Subsequent studies have also insinuated mutations in leucine repeat kinase-2 (LRRK2), Parkin, PTEN-induced putative kinase 1 (PINK1), as well as DJ-1 causing familial forms of PD. This review will attempt to discuss the structure, function, and development in genome editing mediated by CRISP/Cas9 system. Further, it describes the genes involved in the pathogenesis of PD and the pertinent alterations to them. We will pursue this line by delineating the PD linkage studies in which CRISPR system was employed. Finally, we will discuss the pros and cons of CRISPR employment vis-à-vis the process of genome editing in PD patients' iPSCs.

Caspase-7 deficiency in Chinese hamster ovary cells reduces cell proliferation and viability

BACKGROUND: Chinese hamster ovary (CHO) cells are the most commonly used mammalian host cell In the commercial-scale production of biopharmaceutical proteins. Modification of genes involved in apoptosis may improve the productivity of CHO cells. Executive caspases, including caspases 3 and 7, play critical roles in apoptosis. The effects of the ablation of the caspase 7 gene on proliferation and viability of CHO cells remains unknown. In this study, we applied clustered regularly interspaced short palindromic repeat (CRISPR/Cas9) to target caspase 7 gene of CHO K1 cell via all in one and homology targeted integration strategies. Consequently, the effect of caspase 7 deficiency on cell proliferation, viability, and apoptosis was studied by MTT assay and flow cytometry. RESULTS: Findings of gel electrophoresis, western blotting, and sequencing confirmed the caspase 7 gene silencing in CHO cells (CHO-KO). Proliferation assay revealed that caspase 7 deficiency in CHO cells resulted in the reduction of proliferation in various CHO-KO clones. Besides, the disruption of caspase 7 had negative effects on cell viability in exposure with NaBu which confirmed by MTT assay. Results of flow cytometry using Anexin V/PI demonstrated that Nabu treatment (11 mM) declined the percentage of live CHO-K1 and CHO-KO cells to 70.3% and 5.79%. These results verified that the CHO-K1 cells were more resistant to apoptosis than CHO-KO, however most of CHO-KO cells undergone early apoptosis (91.9%) which seems to be a fascinating finding. CONCLUSION: These results reveal that caspase 7 may be involved in the cell cycle progression of CHO cells. Furthermore, it seems that targeting caspase 7 is not the ideal route as it had previously been imagined within the prevention of apoptosis but the relation between caspase 7 deficiency, cell cycle arrest, and the occurrence of early apoptosis will require more investigation.

The effects of specific expression of apoptin under the control of PSES and PSA promoter on cell death and apoptosis of LNCaP cells.

OBJECTIVES: Apoptotic effect of apoptin has been demonstrated in numerous studies. However, its tumor specificity has been questioned by some reports. The aim of this study was to confine the expression of apoptin in the prostate tumor cells by inducing its gene expression under the control of a chimeric enhancer composing of prostate-specific membrane antigen (PSMA) and prostate-specific antigen (PSA) regulatory elements (PSES). Furthermore, we investigated the effects of apoptin expression on LNCaP prostate carcinoma cell survival and apoptosis using MTT assay and annexin V/7-AAD flow cytometry assay. MATERIALS AND METHODS: Recombinant plasmids containing apoptin gene under the control of PSES/PSA promoter or Cytomegalovirus (CMV) promoter were constructed. Tumor cell lines including LNCaP cells and HeLa cells, and LX-2 cells (as a normal control) were transfected with the plasmids and the expression of apoptin was evaluated by real time-PCR and western blot analyses. The effects of apoptin expression on cell survival and apoptosis were then investigated using MTT and annexinV/7-AAD flow cytometry assay, respectively. RESULTS: Western blot and real time PCR analyses confirmed the specific expression of apoptin under the control of PSES/PSA regulatory element in the LNCaP cells, while CMV promoter caused apoptin expression in both tumor and normal cell lines. Apoptin expression significantly increased cell death and apoptosis in tumor cells when compared with the normal cells (P<0.001). CONCLUSION: These results suggest that PSES/PSA regulatory element may be considered as an efficient approach for specific expression of apoptin gene in prostate tumor cells and treatment of prostate cancer.

Expression and purification of TAT-NDRG2 recombinant protein and evaluation of its anti-proliferative effect on LNCaP cell line.

N-myc downstream regulated gene2 (NDRG2) belongs to tumor suppressor protein family of NDRG. Anti-proliferative and anti-metastasis of NDRG2 overexpression has been demonstrated in a number of tumors. The aim of this study was to fuse the gene of Trans Activator of Transcription (TAT) protein transduction domain with NDRG2 gene and express and purify TAT-NDRG2 fusion protein in order to investigate the effects of TAT-NDRG2 protein on proliferation and apoptosis of LNCaP prostate carcinoma cell line. pET28a-TAT-NDRG2 and pET28a-NDRG2 plasmids were constructed and transformed into E. coli-BL21(DE3). TAT-NDRG2 and NDRG2 proteins were expressed in the bacteria, purified using affinity chromatography and verified using western blotting. The effects of TAT-NDRG2 and NDRG2 protein treatment on LNCaP cells proliferation and apoptosis were evaluated using MTT assay and AnnexinV, 7-AAD flow cytometry assay, respectively. Western blot analysis confirmed the expression and purification of TAT-NDRG2 and NDRG2 proteins. Treatment of LNCaP cells with TAT-NDRG2 protein increased cell death and induced apoptosis significantly (P < 0.05) compared to control and NDRG2 protein-treated cells. These results suggest that TAT-NDRG2 protein can be considered as a therapeutic modality for the treatment of tumors.

Assessment of different permeabilization methods of minimizing damage to the adherent cells for detection of intracellular RNA by flow cytometry.

BACKGROUND: Various fixation and permeabilization techniques have been developed for detection of intracellular antigens by flow cytometry; however, there are few studies using flow cytometry to detect the frequency of intracellular nucleic acids, particularly RNA. We tested six different permeabilization methods in order to gain access to a high quality method with minimal damage to intracellular components focusing on 18S rRNA in HeLa cells. METHODS: HeLa cells were fixed in 2% paraformaldehyde. A variety of detergents and enzymes including saponin, TritonX-100, Tween-20, NP40, Proteinase K, and streptolysin O were used to optimize a protocol of permeabilization for the flow cytometric enumeration of intracellular 18S rRNA. Treated cells were subjected to standard protocol of flow cytometric in situ hybridization in the presence of FITC-labeled sense and antisense probes to detect 18S ribosomal RNAs. Samples were then analyzed on a FACSCalibur flow cytometer. To evaluate cell morphology, following hybridization the cells were fixed on glass slide, covered with DAPI, and evaluated on a fluorescent microscope with appropriate filter sets. RESULTS: In comparison with other methods, maximum cell frequency in percentage and fluorescent intensity (M1 = 2.1%, M2 = 97.9%) were obtained when the cells were treated with 0.2% Tween-20 and incubated for 30 min (p = 0.001). CONCLUSION: Our study indicated that the highest levels of mean fluorescence could be obtained when the cells were treated with Tween-20. However, it should be taken into consideration that for a successful flow cytometric result, other interfering factors such as hybridization conditions should also be optimized.