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Methods PowerPoint Presentation

Methods

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Methods

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  1. Production and Transduction of a Recombinant, Lentiviral, Vector, Carrying, EGFP, Gene,Hedyeh Alizadeh¹٭, Dr. Rouzbeh Chegeni², Dr. Mohammadmehdi Akhondi³, Dr. Hamid Reza Khorram Khorshid³ 1. Biology Department, Research and Science Branch of Islamic Azad University, Tehran, Iran 2. Shaheed Beheshti Medical University, Tehran, Iran 3. Reproductive Biotechnology Research Center, Avicenna Research Institute (ACECR), Tehran, Iran Objectives Methods Results Transduction with the 24 hour supernatant demonstrated a better result than 48 hour supernatant. Green color in spermatogonial cells under the UV light after cell passage illustrated the maintenance and expression of EGFP which indicates the entrance of EGFP gene into the cell genome. Lentivector mediated production of carrying EGFP ia a method for transfection of different cells. generation III of these vectors are not replication competent and are pseudotyped with the VSVG protein which will adopt the wide host range of the VSV. lentivectors have the ability to integrate the gene of interest in the host genome and result in permanent expression. EGFP gene was subloned into plenti6v5/dest vector. The transgene and/or the packaging vectors were cloned and purified using JM109 bacterial cells. Calcium phosphate transfection using HEK293FT cell line was performed to produce the EGFP carrying lentivectors. Spermatogonial stem cells were then transduced with the EGFP carrying lentivectors. Conclusions By means of recombinant lentiviral vectors it is possible to integrate the gene of interest into the cell genome. This can lead to the final production of transgenic animals. References