Transfection of CRISPR vector for MTRR (rs1801394) polymorphism in HEK293T cell line
Transfection of CRISPR vector for MTRR (rs1801394) polymorphism in HEK293T cell line
Zahra Momtahan,1Hamzeh Rahimi,2Maryam Eslami,3Elham Tafsiri,4,*
1. Department of Genetics, Faculty of New Sciences and Technologies, Tehran Medical Sciences, Islamic Azad Univeristy, Tehran, Iran 2. Molecular Medicine Department, Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran 3. Department of Genetics, Tehran Medical Sciences, Islamic Azad Univeristy, Tehran, Iran 4. Molecular Medicine Department, Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran
Introduction: The human methionine synthase reductase gene (MSR; gene symbol: MTRR) is a housekeeping gene located on chromosome 5 and consists of 21 exons. MTRR is a family of electron transferases. It has been reported in several studies that the MTRR (rs1801394) polymorphism is one of the single nucleotide polymorphisms associated with many diseases such as autism, schizophrenia, cervical cancer, and meningioma and so on. Therapeutic benefits have been provided to gene therapy, including the modification of genes through disruption, editing, or replacement. Gene therapies are increasingly being used in human patients and non-human models to treat human diseases. One of the recently used gene therapy techniques is the CRISPR-Cas system. Many approaches have been developed and applied in a variety of fundamental research using CRISPR-Cas9 systems. Among the various methods of this technique, knock-in/out, base editing, and prime editing are most promising. The purpose of this study is to transfect the CRISPR vector for the MTRR (rs1801394) polymorphism into HEK293T cell line.
Methods: The sgRNA was designed for the polymorphism MTRR (rs1801394). Our vector was a 3rd generation lentiviral expressing plasmid specialized for base editing-4 and having mCherry as a marker. The NEB strain of transformed bacteria harboring the plasmid was cultured on an LB agar plate. The ampicillin 100 g/ml was our bacterial resistance. The transformed bacteria were cultured in two LB agar plates named plasmid A and plasmid B with different amounts of bacteria from the semi-liquid primary culture medium. We performed the plasmid extraction using the Roche kit. Electrophoresis and also NanoDrop, which examines the purity of our plasmid, have been performed. The HEK293T cell line cultured and counted in Neobar lam. We separately transfected our mCherry and control plasmids, which had GFP as a marker, into HEC293T cells with Lipofectamine 2000. Finally, we observed the transfection results under a fluorescence microscope.
Results: The plasmid plates A and B both grew well. The results of electrophoresis and NanoDrop showed that at a concentration of almost 120 ng of the extracted plasmid, a bright band was generated on the electrophoresis gel, indicating the correct extraction of our plasmid. About 10,000 to 15,000 HEK293T cells were observed in the 96-well plate. In this research, we observed the transfected cells under a fluorescence microscope to ensure that the desired plasmid entered the HEK293T cells. The presence of red fluorescent color under the fluorescence microscope indicated that our plasmids were correctly introduced into the cells. Also those cells transfected separately for the control plasmid containing the GFP marker were also examined. The presence of green fluorescent color under the fluorescence microscope indicated that our control plasmids were also correctly introduced into the cells.
Conclusion: It can be concluded that the base editing method, especially the fourth generation of base editing called BE4max, in the CRISPR-Cas9 technique can be a targeted and potential treatment method for those suffering from MTRR (rs1801394) polymorphism and similar polymorphisms. Furthermore, it was shown that the 3rd generation lentiviral CRISPR plasmids can be a suitable and effective vector in the field of base editing.