Construction Of Mouse Nanog Gene Lentiviral Vector And Its Expression In Mesenchymal Stem Cells

Background:Chambers and Mitsui used different ways to clone the gene of nanog at the same time in 2003. nanog can maintain the pluripotency of embryonic stem cells (ESCs). As we known, the studies have shown that LIF-LIFR/gp130-JAK-STAT3, and OCT-3/4 are the major ways to maintain the pluripotency of ESCs. But nanog can do it by itself; it is the horizontal way to maintain the pluripotency of ESC as STAT3 and OCT-3/4. Mesenchymal stem cells(MSCs)have the characteristics of persistent proliferation and multi-directional differentiation as the stem cells, and there are less ethical dispute in the application of MSCs. MSCs can migrate to a variety of acute injured tissues in vivo, then differentiate into different tissue cells under the control of local microenvironment, and have the characteristics of passing the blood-brain barrier easily and less immunological rejection, so they are the ideal seeds for transplantation as a therapeutic method. In recent years, transplantation of MSCs as a therapeutic method has been the focus of research. Lentiviral vector can infect separated and unseparated cells, transfer comparatively large gene fragment or inlet two different genes into one cell, express objective gene longer, rarely induce the host immune response, or infect the same cell time after time, so it is the good vector for gene therapy.Therefore, this study design to transfer the nanog gene into MSCs mediated by lentiviral vector, and detect the expression of nanog in the MSCs, and provide the experimental evidence for the next research on whether the nanog can effect the differentiation and multiplication of MSCs or effect the pathological and physiological process of other cells.Objective:1. The construction and identification of lentiviral vector plasmid recombined with nanog gene2. Modification of mesenchymal stem cells by nanog gene mediated by lentivirus. 3. Identification of the expression of nanog in the modified mesenchymal stem cells.4. Observing whether nanog has the effect on MSCs in proliferation.Methods:1. Primers were designed according to the nanog gene sequences reported in Genbank and the restriction sites of the vector. The gene fragment of nanog is amplified with PCR.2. The gene fragment of nanog and PNL lentiviral vector plasmid are digested with Sal I and Bam H I restriction enzyme, then joined by T4 DNA joining enzyme. The recombination plasmid pNL-Nanog-IRES2-EGFP is obtained. The recombination plasmid is transformated and the positive clone is chosen and identified with restriction endonuclease and sequencing after screening with antibiotics.3. The constructed transfer plasmid pNL-Nanog-IRES2-EGFP (or pNL-IRES2-EGFP,as mock group) with the packaging plasmid pHELPER and the envelop plasmid pVSVG were co-transfected into 293T cells. The supernatant was collected after transfection and was concentrated by centrifugation. Whether the nanog gene was included in the replication-defective lentiviral vector particles was identified by PCR using the genome extracted from the resulting concentrated lentiviral vectors preparations as template.4. The mouse bone marrow-derived mesenchymal stem cells (MSCs) were separated and cultured with the combination of density gradient centrifugation and adherent culture,and the expressions of CD29,CD34,CD45 in MSCs were detected with flow cytometry.5. The supernatant of lentiviral vector is added into MSCs and cultured. The expression of green fluorescence is observed under fluorescence microscope.6. The expression of mRNA of nanog in nanog-MSCs is detected by RT-PCR, the expression of nanog protein in nanog-MSCs is detected with western blot.7. The shape of cells in nanog-MSCs and pNL-MSCs groups is compared after adding bFGF in the culture , and the proliferation of MSCs is detected by the way of MTT. Results:1. The recombination plasmid pNL-Nanog-IRES2-EGFP is identified with restriction endonuclease and sequencing .The resulting 918bp nanog gene fragment was confirmed .2. The recombination plasmid pNL-Nanog-IRES2-EGFP and the plasmid pNL-IRES2-EGFP are transfected into 293T cells respectively, a great deal of green fluorescence is observed in both of them. The virus titre is 6.0×107TU/mL. The genome of virus is detected by PCR, and the resulting 918bp nanog gene fragment in pNL-Nanog-IRES2-EGFP is confirmed.3. The results of flow cytometry show that the expression of CD34,CD45 in the primary generation of adherent bone marrow cells is weakly positive , then is negative in the following generations. The expression of CD29 is positive in primary generation, and the efficiency of expression is stronger in the following generations. These evidences suggest that the cells obtained are MSCs.4. The green fluorescence can be observed in both pNL-MSCs and nanog-MSCs groups, and not be observed in the MSCs which are not infected。5. The resulting 918bp nanog gene fragment was confirmed by RT-PCR in nanog-MSCs group, not in pNL-MSCs group.6. The results of western blot show that there was a 37KD band in the nanog-MSCs group, and not in the pNL-MSCs and MSCs groups .7. The shape of cells in nanog-MSCs and pNL-MSCs groups was same after adding bFGF in the culture , and there was no significant different on the proliferation rates between two groups.Conclusions:1. The plasmid pNL-Nanog-IRES2-EGFP was successfully constructed with double digestion and gene recombinant.2. High titer lentiviral particles were produced with liposome co-transfected in 293T cells, the nanog gene can be inserted into the virus genome.3. The infection efficiency of obtained lentivirus particles on MSCs was high, the nanog gene can be inserted into the MSCs genome, and be transcried and expressed correctly.4. Whether the nanog gene inserting in the MSCs genome has the effect on the proliferation of MSCs is still unknown.