Composition and method for improving recombination efficiency of transgenic cells

Abstract

The invention discloses a composition and a method for improving recombination efficiency of transgenic cells. Through transgenic cells processed by adopting a culture medium containing SCR7, RS-1 orL755507, the HDR efficiency of the transgenic cells is improved. According to some embodiment of the invention, through using specific sgRNA combination, the higher transgenic efficiency is achieved,and the defects that a fertilized ovum is damaged more seriously because of secondary injection and the positive reproductive number is insufficient because the birth rate is dropped more seriously are solved.

Description

technical field [0001] The invention relates to the field of transgenes, in particular to a composition and a method for improving the recombination efficiency of transgenic cells. Background technique [0002] Animal models, especially transgenic mouse models, are an important tool for various life science researches and have a wide range of applications. [0003] Among many transgenic animals, point mutant mice are common transgenic animal models due to their ease of cultivation and relatively low cost. There are mainly three methods for preparing point mutant mice in the prior art: [0004] The first is the traditional ES targeting scheme. Its disadvantages are: (1) A series of steps such as complex targeting vector construction, ES cell screening, and chimera mouse breeding are required. Large, time-consuming, and low efficiency; (2) Different strains of mice require different ES cells. At present, the common C57BL / 6 strain is used for ES targeting, and no related stra...

AI Technical Summary

Problems solved by technology

[0004] The first is the traditional ES targeting scheme. Its disadvantages are: (1) A series of steps such as complex targeting vector construction, ES cell screening, and chimera mouse breeding are required. Large, time-consuming, and low efficiency; (2) Different strains of mice require different ES cells. At present, the common C57BL / 6 strain is used for ES targeting, and no related strains have been seen for other strains. to report

For example, point mutation models are mainly used to study human diseases, which often require the use of special strains. Since there are no related ES cells, ES targeting technology cannot be used for preparation

[0005] The second is the TALEN scheme: its disadvantages are: (1) The TALEN protein designed to recognize a 20-base sequence contains more than a thousand amino acids, which may cause the body's immune response, which will reduce the role of TALEN in cells; (2) ) TALEN also has off-target problems, which may be caused by the state of chromosomes in cells, such as TALEN technology has no effect on gene sequences in heterochromosomal structures; (3) Is TALEN technology capable of incorporating large fragments? At present, the application of TALEN technology mainly focuses on gene knockout, but whether it is suitable for large fragment gene insertion needs further study

Disadvantages of wild-type hCas9: (1) It is easy to cause off-target effects; (2) It is easy to cause lethality for important mutation points of some important genes

Disadvantages of the mutant Cas9_D10A: Two different sgRNAs recognize the sequences on both sides of the target site separately, and then combine with single-nicking Cas9 nuclease to cut the two strands separately, thereby improving the specificity of target site recognition. Reduced off-target effects, but due to the low efficiency of single-cut Cas9 nuclease, the positive rate of Cas9_D10A scheme is relatively low

The second DNA break repair pathway is homology-mediated repair (HR, homology-directed repair). This repair mechanism based on homologous recombination has high fidelity, but has a low probability of occurrence

[0010] Homozygous lethal transgenic animals, such as transgenic mice, are generally constructed using the method of conditional point mutation of fertilized eggs. The construction process is cumbersome, the construction is very difficult, the positive rate is very low, and the cycle is very long

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