Gene transfer system and method for specific poultry primordial germ cell

Abstract

The invention provides a gene transfer system and method for a specific poultry primordial germ cell. The gene transfer system comprises a transposase helper plasmid containing a vasa promoter with a nucleotide sequence shown in SEQ ID NO.1. The gene transfer method comprises the steps of (1) constructing the transposase helper plasmid containing the vasa promoter or containing the vasa promoter and a universal enhancer element by using a bioengineering method; (2) constructing a transposon donor plasmid containing a target gene expression box required to be transferred by using the bioengineering method; (3) extracting the plasmids, uniformly mixing the transposon donor plasmid with the transposase plasmid according to a ratio of 1 to 2, and adjusting the final concentration of a mixed plasmid to be 500 ng-1.5 mu g / mu L; and (4) wrapping the mixed plasmid obtained in the step (3) with an embedding agent and transfecting PGCs cells in early embryo blood of poultry. Through the gene transfer system and method, efficient specific integration of exogenous genes in PGCs genomes in embryos of the poultry can be realized, so that the purpose of improving preparation efficiency of transgenetic poultry is achieved.

Description

technical field [0001] The invention belongs to the field of animal genetic engineering and relates to the establishment of a gene transfer system specific to poultry primordial germ cells and a poultry transgenic method mediated based on the gene transfer system. Background technique [0002] Transposons are a type of DNA sequence that can freely jump (replicate or translocate) on the genome. It was first discovered in corn by Mc.Clintock in 1940, and later found in various organisms such as bacteria, fungi, and insects. . In recent years, transposon systems have been applied to non-virus-mediated transgenesis, especially in transgenic mammals. However, in the preparation of transgenic poultry, based on the special physiological and reproductive characteristics of poultry, that is: the early development of embryos has already started in the reproductive system of the female poultry, so even the newly laid eggs have developed into about 60,000 morphologically The blastodis...

AI Technical Summary

Problems solved by technology

Commonly used transposon systems include salmon SB (Sleepingbeauty), PB (PiggyBac), Tol2, etc., all of which are transposed according to the "cut-and-paste" method, which can effectively improve the efficiency of transgenic animal production, but the transposon system It does not have germ cell specificity, and its gene transfer rate in somatic cells and germ cells is equivalent, so the transgenic offspring still has problems such as many chimeras and low efficiency of gonad gene transfer

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