Study On The Technology Of Correcting Point Mutation By Specially Designed Oligonucleotides

Objective: The technique that correcting the point mutation by specially designed oligonucleotides(ONs) is a latest molecular biological technique developed in recent years. With this technique, we can correct or introduce a assigned point mutation in any site of genes, which sequence already known. Contrast to the traditional gene recombination method, this technique hasn't any influence on the construction of genome itself and doesn't produce immunoreaction and random integration caused by virus or plasmid vectors. Some genetic diseases, such as hemophilia B , are partly caused by a given point mutation. In this study, we aim to establish a in vitro and in vivo research methods of correcting point mutation with specially designed oligonucleotides, and explore impact factors on nuclear transfection rate and mutation conversion rate. A point mutation was introduced into the normal factorâ…¨gene to study the approachs of elevating the transfection rate and retrieve rate, and provide some experimental data for this technique to be ultimately used in gene therapy of hemophilia B or other diseases caused by point mutation.Methods: This study was divided into three parts. The chemical carrier for the assigned ONs is synthesized in the first part. Through reducing incubation time, the low-level lactosed polyethyleneimine (L-PEI) was prepared by reductive amination of PEI with lactose. The content of galactose residue in the complex was measured with phenol-sulfuric acid method and the lactose rate was calculated. In the second part, transfection experiments in vivo and in vitro are performed. Three types of cell lines: BRL,HL-7702 and SMMC-7721 are respectively transfected with the fluorescently labeled L-PEI-ONs , then the transfection rate was studied with Confocal Scanning Laser Microscope(CSLM). This L-PEI-ONs compound was also injected into the caudate lobe of the SD rat's liver, cell uptake of the ONs in different time points after injection is determined with CSLM. Mutation experiment is the last part of this research.