Cell and Tissue Journal

Abstract Introduction: Homeodomain transcription factor 1 (PDX-1) is necessary for proper pancreatic development and β-cell function. The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) system, in conjunction with CRISPR-associated protein 9 (Cas9), facilitates modifications, particularly deletions, within the genome. The use of lentiviral vectors for gene transfer to somatic stem cells has increased significantly. They harbor advantageous features, including a relatively high gene loading capacity, a diminished likelihood of gene expression silencing following integration into the genome, and high efficiency in delivering genes to a variety of target cells. Furthermore, they elicit no undesirable immune reactions and offer a broad selection of alternative envelope proteins.
Aim: This study aims to introduce a mutation in the chicken PDX-1 gene. A guide RNA (gRNA) will be designed to complement a specific region of the PDX-1 gene. This approach aims to create targeted mutations with potential therapeutic applications in gene therapy.
 
Materials and Methods: The PDX-1 gene was cloned using the LentiCRISPRv2GFP vector. To delete and modify the PDX-1 gene, a specific single guide RNA (sgRNA) sequence is designed. This sgRNA sequence is approximately 100 base pairs (bp) in length, with about 76 bp derived from the scaffold segment already integrated into the LentiCRISPRv2GFP vector.
The region responsible for binding the sgRNA to the target genomic sequence is 20 nucleotides long and is complementary to 20 nucleotides in exon one of the PDX-1 gene. The CRISPR Design Tool (http://crispr.mit.edu) was used to design a specific sgRNA sequence targeting the PDX1 locus (sgPDX1).
The double-stranded oligonucleotide fragment and the LentiCRISPRv2GFP vector were subjected to enzymatic digestion using the BsmbI restriction enzyme. To create the LentiCRISPRv2GFP-sgPDX1 vector, the cleaved sgPDX1 sequence was joined with the cleaved vector. This was accomplished by mixing the vector and nucleotide fragment and subjecting them to a ligation reaction using the T4 ligase enzyme. The resulting vector contains a transcript of the sgRNA, specifically sgPDX1.
The heat shock method was employed to transform Escherichia coli Top10 host cells with the cloned LentiCRISPRv2GFP-sgPDX1 vector. To confirm the presence of the sgPDX-1 gene in the vector, a polymerase chain reaction (PCR) was conducted using primers targeted at a portion of the U6 promoter sequence (forward primer) and a segment downstream of the sgRNA gene region (reverse primer). Additionally, each PCR product was analyzed using enzymatic digestion with EcoRV and BsmBI enzymes. To confirm the successful cloning, the PCR product was sequenced.
Results: The PCR results showed an amplified fragment measuring 267 base pairs (bp), which indicated the presence of the sgPDX-1 gene. This was visualized on an agarose gel. Additionally, the reverse digestion with two restriction enzymes produced a 2000 bp fragment isolated from the vector. The sequencing results confirmed that the cloning process was successful.
Discussion: Genome editing technology has been highlighted in bird species due to their significant potential for economic production and creating models for biological research. The application of CRISPR gene editing technology in avian research has yielded promising advancements, particularly in the modification of somatic cells and tissues in birds. By precisely targeting primordial germ cells (PGCs), researchers can implement specific genetic modifications within the chick genome, thereby facilitating the development of genetically edited chicks. Numerous studies have investigated the role of the PDX-1 gene in gene therapy using the CRISPR/Cas9 system across various organisms. However, this study is the first to design and produce a recombinant construct containing the chicken PDX1 gene using a lentiviral vector with the CRISPR/Cas9 construct. This advancement aims to create a practical research platform for future studies by enabling the direct transfer of this recombinant construct into chick embryos and cells.
Conclusion: The chicken PDX-1 gene was cloned into a lentiviral vector containing the CRISPR/Cas9 construct. The recombinant vector LentiCRISPRv2GFP-sgPDX1 was successfully obtained. The gene construct developed in this study has significant potential for investigating gene function and for use in gene therapy. Consequently, utilizing this construct may enable the creation of an animal model in chicken embryos featuring defects, including an empty organ or tissue cavity. Such a model will allow for the investigation and treatment of the defects using specific drugs and cell lines.

Abstract Aim: In current study, we aimed to reduce Cdc42 gene expression in lung carcinoma related cells, Calu-6, and assayed its effect on cell proliferation.
 Material and Methods: To reduce the expression of Cdc42 gene shRNA system was used and lentiviral system was selected to deliver Cdc42 specific shRNA to Calu-6 cells. Recombinant lentiviruses produced by co-transfection of pMD2G, psPAX2 and p-GFP-C-shLenti plasmids into 293T cells using lipofectamin. Efficiency of transfection and transduction assessed by florescent microscopy. Viability of cells treated by recombinant lentiviruses assessed by MTT assay.
 Results: florescent microscopy showed 80% transfection of 293T cells and high rate of Calu-6 cells transduction. MTT assay results revealed that viability of transduced Calu-6 cells reached to %58 and %40 in compare to control and negative control cells, respectively.
Conclusion: recombinant lentiviruses properly transfer Cdc42-shRNA into Calu-6 cells, leading to reduction of cell proliferation. Silencing of Cdc42 gene expression using lentiviruses is persist and long-term effect which can be under attention for gene therapy of lung cancer.
 

Abstract Aim: The aim of this study was to generate recombinant lentiviruses carrying Nurr1 to express it in human cells. Material and methods: The IRES-EGFP fragment was isolated from the pIRES2-EGFP vector using restriction enzymes BglII/NotI and made blunt-ended using Klenow. The transfer vector PNL-EGFP/CMV/WPREdU3 was digested with NheI/XhoI and made blunt-ended. Finally, the isolated IRES-EGFP fragment was inserted into this lentivirus vector to generate lentivirus construct (I). The human Nurr1 gene was then isolated from the PCMX-NOT vector using BamHI and XhoI and inserted into construct (I) pre-digested with BamHI and SalI. At this step lentivirus construct (II) as our final transfer construct was generated. In order to generate recombinant lentiviruses, we then transfected the HEK-293T cell line with transfer vector plus packaging and envelope vectors. Cell medium full of virus particles was collected and passed through Amicon filters to produce a concentrated virus stock. The stock was ultimately used for transduction of fresh HEK-293T cells. EGFP expression was shown under fluorescent microscope and Nurr1 expression was analyzed using RT-PCR. Results: Enzymatic tests confirmed the correct cloning of the hNurr1gene into the lentivirus backbone. Observation by fluorescent microscopy showed EGFP expression post-transfection and post-transduction. RT-PCR demonstrated Nurr1 expression at both stages. Conclusion: In this study, lentiviruses carrying the human Nurr1 gene were produced and used for transduction of human cell line HEK-293T. The transduced cells successfully expressed the Nurr1 gene.

Abstract Aim: By combining Tet-inducible system and lentivirus vectors, we investigated the induction of Enhanced Green Fluorescent Protein (EGFP) reporter gene in poultry cells. Material and Methods: the EGFP gene was placed under Tet-ON system and its induction was studied in liver cell line LMH. First, we transformed competent bacteria separately with an inducible lentivirus vector carrying EGFP and a second vector carrying Tet transactivator rtTA-M2 and prepared a purified maxi-prep stock of either plasmid DNA. Then, we used DNA-calcium phosphate co-precipitation method to co-transfect LMH cells with both plasmid stocks. In the next step, we added different concentrations of Tet analog doxycycline (DOX) to cell growth medium. Results: Increase of DOX concentration caused elevation of gene expression Within the first  24  hours after post-transfection, the expression of EGFP for 0, 0.01, 0.1 and 1 µg/ml of DOX was 0.4, 6.2, 10.3 and 16% respectively, and in the next 24 hours the expression changed to 6.4, 26, 28.3 and 29.7% respectively. However, treatment with the overdose of DOX caused the cell death. Conclusions: Combination of Tet-inducible system originating from bacteria and recombinant lentivirus vectors designed for gene transfer to human cells can jointly promote transgene expression in poultry cells. The concentration of the inducer can directly influence the level of transgene induction.

Abstract Aim: In this study the efficiency of lentiviral gene transfer and cytomegalovirus promoter mediated overexpression has been investigated. Furthermore, the effect of small molecule-mediated inhibition of epigenetic pathways on gene overexpression has been studied. Materials and Methods: Mouse ES cells were transduced with different doses of lentiviruses  harboring Green Fluorescent Protein (GFP) and the percentage of GFP expressing cells was quantified with flowcytometery. The persistence of GFP expression was assessed after 8 days of transduction. Using embryonic stem (ES) cells transduced with a lentiviral vector harboring pancreatic and duodenal 1 (Pdx1) gene, we studied the influence of 5-azacytidin (5-AZA), DZNep, and BIX01294 small molecules on the gene expression system. Results: Lentiviral mediated gene transfer to ES cells with 10 and 20 multiplicities of infection (MOI) resulted in more than 90 percent transgenesis. However, the expression of transgene showed a dramatic decrease during 8 days of post-transduction. Treatment with 5-AZA leads to a 2.5 folds increase in the transgene expression in a permissive culture medium. DZNep also elevated the transgene expression up to 26.5 and 5.9 folds in pluripotency and permissive media respectively. The expression of endogenous Pdx1 gene also increased following DZNep treated. Conclusion: Lentiviral transduction with MOIs more than 10, is an efficient method for transgenesis of mouse ES cells. However, co-application of this method along with the CMV promoter leads to inactivation of the gene expression in long term. DZNep treatment results in reactivation of transgene expression but also can influence the expression of endogenous genes.  

Abstract Aim: The aims of this study were subcloning of Dj-1(PARK7) gene to lentiviral transfer vector, recombinant lentiviruses production and target cells infection with these viruses.
Material and methods: DJ1 gene obtained from pcDNA-DJ1 vector using restriction enzymes EcoR1 and Xho1. Lentiviral transfer vector was coincidental digested using EcoR1 and Sall enzymes. DJ-1 gene was interred into the lentiviral transfer and upstream of Jred gene using T4-DNA ligase. As DJ1-IRES-Jred sequence was placed in downstream and control of CMV promoter. To production of recombinant lentivruses, the made vector is coincidental transferred into the HEK-293T (Human Embryonic Kidney) cells with two packaging and envelope lentiviral vectors. Produced virus was used for target cells infection.
Results: For integrity of subcloning, enzymatic tests and PCR were used.
Fluorescent microscopy also used to show expression of Jred reporter gene that showed the success of our gene transferring .Then RT-PCR was done for showing overexpression of  DJ1 gene in transduced cells compare with normal cells.
Conclusion: This study show the lentiviral vectors success in gene transferring to eukaryotic cells and clear that this vectors can be used in treatment of nervous system diseases.

Abstract Aim: The aim of this study is to examine the efficiency of DNA absorption and gene transfusion to chicken hepatoma cells line LMH at both transfection and transduction levels using recombinant lentiviruses. Then transgene expression level was studied.
Material and Methods: The cells were cultivated in 96-well plates for viral transfection or transduction and then studied using fluorescent microscope in different stages. Green Fluorescent Protein-positive (GFP) cells counted by Grid Cell Counter software and transgen expression level calculated using ImageJ software. Data analyzed using SPSS.
Results: GFP gene expression began 6 and 9 hours after transfection in HEK and LMH cells, respectively. These times were increased, respectively, to 22 and 36 hours post-transduction. Counting GFp < sup>+ cells 48 hours post-transfection showed that HEK and LMH cells have received and expressed 40% and 35% of DNA. These levels increased to 95% and 48% respectively in 72 hours. Also counting HEK⁺ cells at 72 and 96 hours viral post-transduction confirmed the similar pattern of receiving DNA by both HEK and LMH cells. Therefore, analyzing of GFP expression level showed that HEK and LMH classes expressed GFP in 74% and 89% (p < 0.01 significant).
Conclusion: Overall, our results indicated that HEK cells have higher potential to absorption of foreign genes whereas the intensity of expression is higher in LMH cells.