A Lentiviral Vector Expressing Desired Gene Only in Transduced Cells: An Approach for Suicide Gene Therapy

Mohammadi, Zahra; Shariati, Laleh; Khanahmad, Hossein; Kolahdouz, Mahsa; Kianpoor, Fariborz; Ghanbari, Jahan Afrooz; Hejazi, Zahra; Salehi, Mansoor; Nikpour, Parvaneh; Tabatabaiefar, Mohammad Amin

doi:10.1007/s12033-015-9872-3

Cited by 11 publications

(15 citation statements)

References 28 publications

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“…We demonstrated here evidence suggesting that the GFP gene ( EGFP ) can be expressed in promoterless cassettes. During an investigation, we designed and constructed a retroviral transfer vector for the purpose of expressing GFP in transduced cells and with no expression in packaging cells (20). In the production of lentivirus, we expected the GFP not to be expressed in the packaging cells due to the absence of any promoter in the upstream of GFP .…”

Section: Discussionmentioning

confidence: 99%

“…The existence of a cryptic promoter has been proven for some genes (171819). In previous studies, as we were involved in designing and constructing a viral transfer vector which was supposed to express GFP in transduced cell line but not in packaging HEK293T cells, after transfection of the cells, we unexpectedly noticed that the promoterless GFP cassette was expressed (2021). Therefore, the present study was launched to investigate the reason behind the observation.…”

Section: Introductionmentioning

confidence: 99%

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Evidence for expression of promoterless GFP cassette: Is GFP an ideal reporter gene in biotechnology science?

et al. 2019

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Green fluorescent protein (GFP) has played an important role in biochemistry and cell biology as a reporter gene. It has been used to assess the potency of promoters for recombinant protein production. This investigation reveals evidences suggesting that the gfp GFP gene ( EGFP ) could be expressed without the promoter. In a study, a pLenti-F/GFP vector was constructed with the purpose to allow GFP expression in transduced cells but not in packaging cells; however, after transfecting the HEK293T cell line, GFP gene was expressed, compared to pLOX/CW gfp -transfected cells showed expression lag, lower levels and reduced percentage of GFP expression in the cells. This unexpected result could be due to auto transduction in packaging cell, possible retrotransposon activity in the cell line, possible contamination of pLenti-F/GFP with the pLOX/CWgfp and possible presence of a promoter within backbone of the vector. All the possibilities were ruled out. To exclude the possibility that a sequence within the region might act as a promoter, the fragment to be transfected was minimized to a region containing “from the start of the GFP gene to 5’LTR R”. The GFP gene was again expressed. Therefore, our findings suggest the EGFP does not need promoter for expression. This should appeal to the researchers designing GFP based assays to evaluate the potency of promoters, since possible aberrant expression may have a potential to influence on the results of a planned experiment.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Evidence for expression of promoterless GFP cassette: Is GFP an ideal reporter gene in biotechnology science?

et al. 2019

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“…According to jumping in the lentivirus life cycle the Splicing trick for preferential gene expression F Pourzadegan et al construct is able to express the GFP in transduced cells but not in packaging cells. 18 The present study introduces a novel method to circumvent this problem by taking advantage of lentiviral life cycle and intron-splicing machinery in mammalian cells. In this regard, we have inserted the intron of beta globin between the CMV promoter and the gfp gene (a reporter gene instead of a toxin) in the reverse direction.…”

Section: Discussionmentioning

confidence: 99%

“…According to our previous attempt, 18 and based upon the basic rules of molecular biology and recruitment of the retrovirus life cycle, 16,17 we constructed a lentiviral DEST plasmid expressing the target gene in transduced cells but not in packaging cells. To monitor gene expression within the cells and to investigate our hypothesis, and of course for convenience, the green fluorescent protein (GFP) reporter gene was used as a substitute of the toxic gene.…”

Section: Introductionmentioning

confidence: 99%

Using intron splicing trick for preferential gene expression in transduced cells: an approach for suicide gene therapy

et al. 2015

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Suicide gene therapy is one of the most innovative approaches in which a potential toxic gene is delivered to the targeted cancer cell by different target delivery methods. We constructed a transfer vector to express green fluorescent protein (GFP) in transduced cells but not in packaging cells. We placed gfp under the control of the cytomegalovirus (CMV) promoter, which is positioned between the two long-terminal repeats in reverse direction. The intron-2 sequence of the human beta globin gene with two poly-A signals and several stop codons on the antisense strand was placed on the leading strand between the CMV promoter and gfp. For lentiviral production, the HEK293T and line were co-transfected with the PMD2G, psPAX2 and pLentiGFP-Ins2 plasmids. The HEK293T and line were transduced with this virus. PCR was performed for evaluation of intron splicing in transduced cells. The GFP expression was seen in 65% of the cells transduced. The PCR amplification of the genomic DNA of transduced cells confirmed the splicing of intron 2. The strategy is significant to accomplish our goal for preserving the packaging cells from the toxic gene expression during viral assembly and the resultant reduction in viral titration. Also it serves to address several other issues in the gene therapy.

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“…Lentivirus supernatants were harvested for two or three times, every 12 h, concentrated by ultracentrifuge at 47,000×g for 2 h at 4 °C. Lentivirus titer examined by flow cytometry analysis of reporter positive 293 T cells (Attune Acoustic Focusing Flow Cytometer, ABI, FlowJo software) 48 .…”

Section: Plasmids Viral Vectors Construction and Luciferase Assay Fmentioning

confidence: 99%

Involved microRNAs in alternative polyadenylation intervene in breast cancer via regulation of cleavage factor “CFIm25”

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Shokri

Rad

et al. 2020

Sci Rep

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Cleavage factor "CFIm25", as a key repressor at proximal poly (A) site, negatively correlates to cell proliferation and tumorigenicity in various cancers. Hence, understanding CFIm25 mechanism of action in breast cancer would be a great benefit. To this aim four steps were designed. First, potential miRNAs that target 3′-UtR of CFIm25 mRnA, retrieved from targetscan web server. Second, screened miRNAs were profiled in 100 breast cancer and 100 normal adjacent samples. Third, miRNAs that their expression was inversely correlated to the CFIm25, overexpressed in MDA-MB-231 cell line, and their effect on proliferation and migration monitored via MTT and wound healing assays, respectively. fourth, interaction of miRNAs of interest with 3′-UtR of CFIm25 confirmed via luciferase assay and western blot. our results indicate that CFIm25 considerably down-regulates in human breast cancer tissue. qRT-PCR assay, luciferase test, and western blotting confirm that CFIm25 itself could be directly regulated by oncomiRs such as miR-23,-24,-27,-135,-182 and-374. Besides, according to Mtt and wound healing assays of cell lines, CFIm25 knockdown intensifies cell growth, proliferation and migration. Our results also confirm indirect impact of CFIm25 on regulation of mRNA's 3′-UtR length, which then control corresponding miRNAs' action. miRNAs directly control CFIm25 expression level, which then tunes expression of the oncogenes and tumor proliferation. Therefore, regulation of CFIm25 expression level via miRNAs is expected to improve treatment responses in breast cancer. Abbreviations CPSF Cleavage and polyadenylation specificity factor CstF Cleavage stimulation factor PAS Polyadenylation site 3′-UTR 3′-Untranslated region UTR-APA Untranslated region-alternative polyadenylation miRNA/miR MicroRNA CFIm25 Cleavage factor Im 25 RF Random forest classifiers shRNA Short hairpin RNA HCC Hepatocellular carcinoma Living organisms apply complicated mechanisms of gene regulation to generate different cell types, which result in various behaviors from a single genome 1,2. Dynamic and highly polymorphic nature of mRNA polyadenylation, as a functional aspect of gene regulation, is one of the most recent discoveries in this field 3,4. Cleavage and polyadenylation of nascent mRNA accomplishes via application of two large multimeric complexes, which named as cleavage-polyadenylation specificity factor (CPSF) and cleavage stimulation factor (CstF), respectively. The

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A Lentiviral Vector Expressing Desired Gene Only in Transduced Cells: An Approach for Suicide Gene Therapy

Cited by 11 publications

References 28 publications

Evidence for expression of promoterless GFP cassette: Is GFP an ideal reporter gene in biotechnology science?

Evidence for expression of promoterless GFP cassette: Is GFP an ideal reporter gene in biotechnology science?

Using intron splicing trick for preferential gene expression in transduced cells: an approach for suicide gene therapy

Involved microRNAs in alternative polyadenylation intervene in breast cancer via regulation of cleavage factor “CFIm25”

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