A method for detecting low-efficiency genome editing based on polyacrylamide gel electrophoresis and its application
A gene editing and detection method technology, applied in biochemical equipment and methods, microbial determination/inspection, etc., can solve the problems of inability to detect, large workload, low sensitivity, etc., to ensure detection accuracy, reduce detection costs, The effect of simplifying experimental operations
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0061] Example 1. Sensitivity of Genome Editing Efficiency Detection by PAGE Method and Cel I Endonuclease Method
[0062] This example aims to simulate the result of artificial endonuclease knockout gene through the naturally occurring homozygous wild type fragment and small fragment homozygous deletion type fragment, and through the homozygous wild type fragment and small fragment homozygous deletion type Quantitative detection of the PCR product of the fragment and quantitative gradient dilution of the PCR product of the homozygous deletion type fragment of the small fragment, established a model of different knockout efficiencies of the artificial endonuclease ( figure 1 ), to quantitatively compare the sensitivity of the PAGE method and the current general-purpose Cel I endonuclease method to detect genome editing efficiency. Specific steps are as follows:
[0063] 1. Simulate gene editing to obtain gene-edited DNA
[0064] In this example, homozygous wild-type fragment...
Embodiment 2
[0102] Example 2, Application of polyacrylamide gel electrophoresis in detecting the editing efficiency of ZFN
[0103] Since ZFN has been developed for a long time, the technology is relatively mature, and the editing efficiency is low, it is more convincing to use ZFN to verify the method in the above-mentioned Example 1. The following uses it as a tool for gene editing in this example to verify the present invention Methods. In addition, animals with mutations in the MSTN gene will show "double-muscle" traits. In this example, ZFN is used to edit the MSTN gene of sheep fibroblasts to obtain sheep fibroblasts with MSTN gene knockout. Transplantation and embryo transplantation are also of great significance for cultivating good materials for sheep breeds with "double muscle" traits. Specific steps are as follows:
[0104] 1. Obtaining DNA after gene editing
[0105] 1. Preparation of ZFNs
[0106] According to the sequence of sheep (Ovis aries) myostatin (MSTN) gene (GenB...
Embodiment 3
[0135] Example 3. Application of polyacrylamide gel electrophoresis in screening target gene editing clones
[0136] 1. Preparation of monoclonal cells
[0137] Monoclonal cells were prepared by the limiting dilution method, and the specific steps were as follows: the fibroblasts obtained in Step 1 of Example 2 and 3 were further cultured for 72 hours, and the cells were digested with trypsin and counted, and each 10 cm culture dish Add 500 cells and 10mL DMEM culture solution containing 20% fetal bovine serum and 1% double antibody, gently shake the culture dish to distribute the cells evenly, and then place it at 37°C, 5% CO 2 Cultivate in the incubator for 7-10 days; after the monoclonal growth of the cells, digest the cells with a cloning ring and preheated trypsin, transfer them to a 96-well plate for expansion and culture, and then transfer them to a 48-well plate for continued culture; wait until the cells are covered At 80% of the bottom of the 48-well plate, the ce...
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More