Construction of expressing vectors including melanoma differentiation-associated gene-7 (mda-7) fused with the RGD sequences for better tumor targeting
Objective(s): Up to now, many researches have been performed to improve the antitumoral effect of melanoma differentiation-associated gene-7 (mda-7) protein. The purpose of our research was to construct 3 expression vectors producing mda-7 in fusion with RGD (Arginine-Glycine-Aspartic acid) peptide...
| Main Authors: | , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Mashhad University of Medical Sciences
2015-08-01
|
| Series: | Iranian Journal of Basic Medical Sciences |
| Subjects: | |
| Online Access: | http://ijbms.mums.ac.ir/pdf_4729_a18967cb67f59481f0c7af90d9d12829.html |
| _version_ | 1828272437364523008 |
|---|---|
| author | Mahboobeh Khodadad Seyed Younes Hosseini Fatemeh Shenavar Nasrollah Erfani Samaneh Bina Shahin Ahmadian Mohammad-Reza Fattahi Reza Hajhosseini |
| author_facet | Mahboobeh Khodadad Seyed Younes Hosseini Fatemeh Shenavar Nasrollah Erfani Samaneh Bina Shahin Ahmadian Mohammad-Reza Fattahi Reza Hajhosseini |
| author_sort | Mahboobeh Khodadad |
| collection | DOAJ |
| description | Objective(s): Up to now, many researches have been performed to improve the antitumoral effect of melanoma differentiation-associated gene-7 (mda-7) protein. The purpose of our research was to construct 3 expression vectors producing mda-7 in fusion with RGD (Arginine-Glycine-Aspartic acid) peptide and evaluate their expression.
Materials and Methods: mda-7 gene with two different RGD sequences was amplified by PCR then was cloned by TA–cloning system. The colonies including these genes were selected by blue–white screening, colony PCR, and sequencing, respectively. Afterward, the genes were sub-cloned into the expression vector following confirmation by colony PCR and sequencing. In addition, these constructs were transfected into 293 and Huh-7 cells for further expression analysis. The mda-7 gene expression was evaluated by RT-PCR and IF (immunofluorescence assay). DNA laddering test and trypan blue exclusion assays were performed to screen cytotoxicity of prepared plasmids.
Results: Three different mda-7 genes with terminal RGD peptide were cloned correctly into the expression vectors and their expression was confirmed to be suitable by RT-PCR and IF assay. It was shown that expressions were limited to those transfected, GFP shining cells. No significant cytotoxicity was observed by simple assays in all plasmid treated cells. In expressing cells, all forms of mda-7 protein were localized mainly around ER prenuclear compartment while GFP protein was distributed evenly among them.
Conclusion: Theoretically RGD tagged mda-7 would be able to induce apoptosis with more specificity and stronger than the standard one, therefore, these new constructs may have the potential for further researches. |
| first_indexed | 2024-04-13T06:07:40Z |
| format | Article |
| id | doaj.art-11b6afe4d37444e393d5d32cd5c91b1c |
| institution | Directory Open Access Journal |
| issn | 2008-3866 2008-3874 |
| language | English |
| last_indexed | 2024-04-13T06:07:40Z |
| publishDate | 2015-08-01 |
| publisher | Mashhad University of Medical Sciences |
| record_format | Article |
| series | Iranian Journal of Basic Medical Sciences |
| spelling | doaj.art-11b6afe4d37444e393d5d32cd5c91b1c2022-12-22T02:59:12ZengMashhad University of Medical SciencesIranian Journal of Basic Medical Sciences2008-38662008-38742015-08-011887807874729Construction of expressing vectors including melanoma differentiation-associated gene-7 (mda-7) fused with the RGD sequences for better tumor targetingMahboobeh Khodadad0Seyed Younes Hosseini1Fatemeh Shenavar2Nasrollah Erfani3Samaneh Bina4Shahin Ahmadian5Mohammad-Reza Fattahi6Reza Hajhosseini7Gastroenterohepatology Research Center, Shiraz University of Medical Sciences, Shiraz, IranGastroenterohepatology Research Center, Shiraz University of Medical Sciences, Shiraz, IranGastroenterohepatology Research Center, Shiraz University of Medical Sciences, Shiraz, IranCancer Immunology Research Group, Shiraz Institute for Cancer Research, School of Medicine, Shiraz University of Medical Sciences, Shiraz, IranGastroenterohepatology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran|Sciences Institute of Biochemistry and Biophysics, University of Tehran, Tehran, IranSciences Institute of Biochemistry and Biophysics, University of Tehran, Tehran, IranGastroenterohepatology Research Center, Shiraz University of Medical Sciences, Shiraz, IranDepartment Of Biochemistry, Payame Noor University, Tehran Shargh Branch, Tehran, IranObjective(s): Up to now, many researches have been performed to improve the antitumoral effect of melanoma differentiation-associated gene-7 (mda-7) protein. The purpose of our research was to construct 3 expression vectors producing mda-7 in fusion with RGD (Arginine-Glycine-Aspartic acid) peptide and evaluate their expression. Materials and Methods: mda-7 gene with two different RGD sequences was amplified by PCR then was cloned by TA–cloning system. The colonies including these genes were selected by blue–white screening, colony PCR, and sequencing, respectively. Afterward, the genes were sub-cloned into the expression vector following confirmation by colony PCR and sequencing. In addition, these constructs were transfected into 293 and Huh-7 cells for further expression analysis. The mda-7 gene expression was evaluated by RT-PCR and IF (immunofluorescence assay). DNA laddering test and trypan blue exclusion assays were performed to screen cytotoxicity of prepared plasmids. Results: Three different mda-7 genes with terminal RGD peptide were cloned correctly into the expression vectors and their expression was confirmed to be suitable by RT-PCR and IF assay. It was shown that expressions were limited to those transfected, GFP shining cells. No significant cytotoxicity was observed by simple assays in all plasmid treated cells. In expressing cells, all forms of mda-7 protein were localized mainly around ER prenuclear compartment while GFP protein was distributed evenly among them. Conclusion: Theoretically RGD tagged mda-7 would be able to induce apoptosis with more specificity and stronger than the standard one, therefore, these new constructs may have the potential for further researches.http://ijbms.mums.ac.ir/pdf_4729_a18967cb67f59481f0c7af90d9d12829.htmlMda-7RGD peptideTumor targeting |
| spellingShingle | Mahboobeh Khodadad Seyed Younes Hosseini Fatemeh Shenavar Nasrollah Erfani Samaneh Bina Shahin Ahmadian Mohammad-Reza Fattahi Reza Hajhosseini Construction of expressing vectors including melanoma differentiation-associated gene-7 (mda-7) fused with the RGD sequences for better tumor targeting Iranian Journal of Basic Medical Sciences Mda-7 RGD peptide Tumor targeting |
| title | Construction of expressing vectors including melanoma differentiation-associated gene-7 (mda-7) fused with the RGD sequences for better tumor targeting |
| title_full | Construction of expressing vectors including melanoma differentiation-associated gene-7 (mda-7) fused with the RGD sequences for better tumor targeting |
| title_fullStr | Construction of expressing vectors including melanoma differentiation-associated gene-7 (mda-7) fused with the RGD sequences for better tumor targeting |
| title_full_unstemmed | Construction of expressing vectors including melanoma differentiation-associated gene-7 (mda-7) fused with the RGD sequences for better tumor targeting |
| title_short | Construction of expressing vectors including melanoma differentiation-associated gene-7 (mda-7) fused with the RGD sequences for better tumor targeting |
| title_sort | construction of expressing vectors including melanoma differentiation associated gene 7 mda 7 fused with the rgd sequences for better tumor targeting |
| topic | Mda-7 RGD peptide Tumor targeting |
| url | http://ijbms.mums.ac.ir/pdf_4729_a18967cb67f59481f0c7af90d9d12829.html |
| work_keys_str_mv | AT mahboobehkhodadad constructionofexpressingvectorsincludingmelanomadifferentiationassociatedgene7mda7fusedwiththergdsequencesforbettertumortargeting AT seyedyouneshosseini constructionofexpressingvectorsincludingmelanomadifferentiationassociatedgene7mda7fusedwiththergdsequencesforbettertumortargeting AT fatemehshenavar constructionofexpressingvectorsincludingmelanomadifferentiationassociatedgene7mda7fusedwiththergdsequencesforbettertumortargeting AT nasrollaherfani constructionofexpressingvectorsincludingmelanomadifferentiationassociatedgene7mda7fusedwiththergdsequencesforbettertumortargeting AT samanehbina constructionofexpressingvectorsincludingmelanomadifferentiationassociatedgene7mda7fusedwiththergdsequencesforbettertumortargeting AT shahinahmadian constructionofexpressingvectorsincludingmelanomadifferentiationassociatedgene7mda7fusedwiththergdsequencesforbettertumortargeting AT mohammadrezafattahi constructionofexpressingvectorsincludingmelanomadifferentiationassociatedgene7mda7fusedwiththergdsequencesforbettertumortargeting AT rezahajhosseini constructionofexpressingvectorsincludingmelanomadifferentiationassociatedgene7mda7fusedwiththergdsequencesforbettertumortargeting |