Construction method of VDR gene conditionality knockout floxed mouse model

Abstract

The invention belongs to the technical field of biology, and relates to a construction method of a VDR gene conditionality knockout floxed mouse model. According to the method, a gene targeting technique and an ES cell recombination technique are utilized, an Frt-neo-Frt-loxP sequence and an loxP sequence are respectively put on two sides of a VDR gene exon 3 of a bacterium artificial chromosome,a VDR-floxed carrier is constructed, and ES cells are guided through electric shock; after positive ES cells screened out through drug-resistant genes are cloned, through sequencing determination withPCR and a product thereof, the positive ES cells are transplanted in the body of a surrogacy female mouse, and a chimera mouse containing ES cell sources is produced; a male chimera mouse and a female wild type mouse intercross to generate an F1-generation mouse, and through sequencing confirmation with the PCR and the product thereof, a VDR-floxed heterozygote mouse containing Neo genes is obtained; and then the VDR-floxed heterozygote mouse containing Neo genes and an Flp mouse hybridize so that a VDR-floxed heterozygote mouse without neo genes is obtained. Through experimental verification, the VDR-floxed mouse model constructed by the construction method can lead to knockout of a VDR gene exon 3 region, and lead to deletion of VDR expression.

Description

technical field [0001] The invention belongs to the field of biological technology, relates to animal genetic engineering and genetic modification methods, in particular to a Cre-loxp system-based VDR gene conditional knockout floxed mouse model construction method. Background technique [0002] The prior art discloses that vitamin D is a general term for a group of fat-soluble ring-opening steroid substances, and there are two main forms of vitamin D: vitamin D2 (ergocalciferol) and vitamin D3 (cholecalciferol). Vitamin D in the human body mainly comes from the skin, and only 10-20% comes from food intake; 7-dehydrocholesterol and 8-dehydrocholesterol in the skin are converted into vitamin D3 under the irradiation of ultraviolet rays, and vitamin D3 first It is transformed into 25-hydroxyvitamin D3[25(OH)D3] through the action of 25 hydroxylase in the liver, and then through the action of 1α hydroxylase in the proximal tubule of the kidney to generate biologically active 1,...

AI Technical Summary

Problems solved by technology

The traditional gene knockout animal is to completely knock out the target gene in the individual animal, so that the expression activity of the gene in various tissues in the body will be lost from the embryonic stage. Practice shows that this kind of animal model may cause the animal to die in the early embryo Or the abnormality of later development is not conducive to the analysis of the function of the gene in a specific tissue or a specific developmental time, for example, VDR gene knockout homozygous mice (VDR-KO) have hypocalcemia and high PTH after birth, manifested as Abnormal development of rickets, hair loss, reproductive immunity, and nervous system, etc., will die if not corrected by a high-calcium and high-vitamin D diet after weaning; in addition, because VDR is expressed in various tissues, the use of VDR gene knockout mice to study The function of local VDR, due to the complexity of the phenotype, it is difficult to judge whether the effect is in situ or secondary, which leads to wrong conclusions; therefore, the current research practice urgently needs to construct a tissue-specific gene knockout animal model

[0006] Based on the status quo of the existing technology, this application intends to provide a method for constructing a floxed mouse model with conditional knockout of the VDR gene in view of the wide range of physiological functions of the VDR gene, the local mechanism of action is unknown, and the lack of relevant animal research models. VDR-floxed mice that will benefit research practice in the field

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