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A mutant gene and its use for constructing a minipig model of ichthyosis variegata

A technology of mutant gene and miniature pig, applied in the field of genetic engineering

Active Publication Date: 2021-11-19
INST OF ZOOLOGY CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The skin structure of mice is different from that of humans, so there are some limitations in studying disease pathology

Method used

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  • A mutant gene and its use for constructing a minipig model of ichthyosis variegata
  • A mutant gene and its use for constructing a minipig model of ichthyosis variegata
  • A mutant gene and its use for constructing a minipig model of ichthyosis variegata

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Embodiment 1 The establishment of human ichthyosis Bama miniature pig genetic family

[0048] 1) ENU was injected into 15 Guangxi Bama miniature pig boars, the injection dose was 65 mg / kg, and the injection frequency was once a week for three consecutive weeks.

[0049] 2) Semen quality testing. After the mutation, boar semen is tested for quality. The test indicators include boar semen volume, sperm density, sperm survival rate and deformity rate. G1 generation.

[0050] 3) A total of 147 male minipigs of the G1 generation described in step 2) were mated with wild-type female miniature pigs of the same species to obtain G2 generation miniature pigs;

[0051] 4) mating the G1 generation miniature pig described in step 2) with the G2 generation sow described in step 3) to obtain the G3 generation miniature pig,

[0052] 5) Perform phenotypic screening of the G3 generation minipigs described in step 4), and obtain family miniature pigs with a mottled ichthyosis phenot...

Embodiment 2

[0057] Example 2 Gene mutation localization of human ichthyosis Bama miniature pig genetic family

[0058] Through genetic linkage analysis and gene cloning sequencing of G3 pigs to determine the mutation position, extract the DNA of the ear tissue of G3 pigs, and design primers for PCR reaction, electrophoresis separation of PCR products, and obtain the closely linked ABCA12 gene locus mutation site.

[0059] 1. DNA extraction and quality testing

[0060] ①Put an appropriate amount of ear tissue into a 1.5ml centrifuge tube, cut it into pieces with small scissors, add 500 μL of SNET solution and 10 μL of proteinase K, and digest in a shaker at 55°C and 200rmp overnight;

[0061] ②After the tissue digestion is complete, centrifuge at 12000rmp for 1min to precipitate the hair, and transfer the supernatant to a new 1.5ml EP tube;

[0062] ③ Add 500 μL of phenol: chloroform: isoamyl alcohol = 25:24:1, shake at room temperature for 30 minutes;

[0063] ④ Centrifuge the soluti...

Embodiment 3

[0087] Example 3 Characteristic analysis of human ichthyosis Bama miniature pig genetic family

[0088] The phenotype screening of family minipigs with mottled ichthyosis phenotype of the present invention is realized through phenotype observation, skin permeability assay (Skin permeability assay) and transepidermal water loss test (TEWL) measurement.

[0089] The results of phenotypic analysis showed that the model pigs with ichthyosis versicolor had dry, hard, cracked skin and had ectropion phenotype.

[0090] The results of paraffin sections of the skin are shown in Figure 3. Figure 3A It is the skin phenotype of wild-type Guangxi Bama miniature pigs; Figure 3B It is the skin phenotype of the human ichthyosis parma minipig model of the present invention. It was shown that the stratum corneum of the Bama minipig model of human ichthyosis versicolor was obviously thickened, and the basal layer was disordered.

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Abstract

The invention provides an ABCA12 mutant gene of miniature pigs. Compared with the ABCA12 gene of wild-type pigs, the mutant gene has a mutation of IVS49‑727A>G. The invention provides a construct comprising the ABCA12 mutant gene. The present invention also provides a recombinant cell obtained by transforming a recipient cell with the construct. The invention provides a method for preparing a minipig model of human ichthyosis ichthyosis, the method comprising: changing the ABCA12 gene of the minipig, changing the 727th base located in the 49th intron of the ABCA12 gene from A to for G. The ABCA12 mutant gene provided by the invention is used to prepare a large-scale human ichthyosis pig model and to study the pathogenesis of human ichthyosis, which has great guiding significance for clinical prevention, diagnosis and treatment of human ichthyosis.

Description

technical field [0001] The invention belongs to the field of genetic engineering, in particular to an ABCA12 mutant gene. The present invention also relates to the use of the mutant gene for constructing the spotted ichthyosis minipig model and a method for constructing the spotted ichthyosis minipig model. Background technique [0002] Guangxi Bama miniature pigs have stable heredity, good consistency in phenotypic characteristics, relatively mild temperament, small body size, and various organ systems are very similar to the human body, so they are suitable for medical and biological experiments. [0003] Harlequin ichthyosis is an inherited skin disease and the most severe form of autosomal recessive congenital ichthyosis. Affected neonates are born with increased thickness of the stratum corneum layer of the skin, covered with armor-like scales, and are often accompanied by abnormal facial features, including eversion of the eyelids, everted lips, and deformed ears and ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/12C12N5/10C12Q1/6888A01K67/027
CPCA01K67/0278A01K2207/15A01K2217/072A01K2227/108A01K2267/03C07K14/47C12N2510/00C12Q1/6888C12Q2600/156
Inventor 赵建国王霄曹春伟海棠周琪王红梅张颖贾启涛郑千涛
Owner INST OF ZOOLOGY CHINESE ACAD OF SCI
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