New Technique for Producing Hybridoma and Gene Transfection Using Laser Iradiation

激光辐射细胞融合：我们开发了一种利用微加工激光装置在显微镜下诱导细胞融合的新方法。用激光束照射靶细胞进行捕获，将捕获的细胞与激光束固定的细胞接触。脉冲激光束聚焦在接触面上。小鼠骨髓瘤细胞融合率为38%。骨髓瘤细胞和淋巴细胞的杂交瘤发生率为2%。我们证实了它的增殖和IgG的产生。该方法的特点是产生目标细胞的杂交瘤，细胞毒性低，杂交瘤产生率高。激光辐照基因转染：研制了激光转染系统。激光转染的原理是通过脉冲激光照射在细胞膜上打一个小孔，将培养基中含有的基因转移到细胞质中。转染编码增强型绿色荧光蛋白和新霉素磷酸转移酶的质粒，检测其表达。转染率约为10%。用这种方法建立了转化细胞系。该方法不需要任何病毒或特异性载体，可以将基因转染到目标细胞中。

Cell fusion by laser radiation : We developed a new method for inducing cell fusion under microscope using a microprocessing laser device. The target cells were irradiated with laser beams for trapping and trapped cells were placed in contact with the cells fixed by laser beam. The pulse laser beams are focused on the contact surface. The fusion rate of mouse myeloma cells was 38%. Rate of hybridoma production of myeloma cell and lymphocyte was 2%. We confirmed its proliferation and the production of IgG.This method is characterized by production of hybtidomas of target cells, lower cell toxicity and a high rate of hybrid production.Gene transfection by laser radiation : We developed the transfection system using laser beams. The principle of transfection by laser is that a small hole is made in the cell membrane by pulse laser irradiation and genes containing in the culture medium is transferred into the cytoplasm. We transfected plasmids encoding enhanced green fluorescent protein and neomycin phosphotransferase, and detected the its expression. The transfection rate was approximately 10%. In this method we established the transformed cell line. This method didn't need any virus nor specific vector and enables us to transfect gene to targeted cells.