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Method for constructing human epidermolytic palmoplantar keratoderma mouse model

A technology of epidermolysis and palmoplantar keratosis, which can be applied to other methods of inserting foreign genetic material, using micro-injection, using vectors to introduce foreign genetic material, etc., and can solve problems such as the complexity of the generation mechanism

Inactive Publication Date: 2015-05-06
ZHEJIANG UNIV
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  • Description
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  • Application Information

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Problems solved by technology

Therefore, the generation mechanism of indels may be more complex, and its significance in disease occurrence is far greater than that of DNA point mutations

Method used

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  • Method for constructing human epidermolytic palmoplantar keratoderma mouse model
  • Method for constructing human epidermolytic palmoplantar keratoderma mouse model
  • Method for constructing human epidermolytic palmoplantar keratoderma mouse model

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Embodiment 1

[0045] Embodiment 1 Contains the construction of the targeting vector plasmid of recombinant Krt9 gene

[0046] 1. The Krt9 gene was synthesized by three-segment PCR, and cloned into the PL253 vector by enzyme digestion and ligation

[0047] (1) Three pairs of PCR primers were designed, using the Krt9-BAC plasmid (purchased from CHORI Institute) as a template, and the cloning of the Krt9 gene (Krt9-PCR1 and Krt9-PCR2, Krt9-PCR3) was completed step by step (Table 1). The three-fragment PCR method uses PrimerStar high-fidelity enzyme, and the system is as follows (50 μl):

[0048]

[0049] The reaction program was denaturation at 94°C for 5 min, followed by sub-cycles, each cycle was denaturation at 94°C for 30 s, annealing at an appropriate annealing temperature for 30 s, extension at 72°C for 3 min, and finally extension at 72°C for 5 min. The PCR amplification products were electrophoresed on 1.5% agarose gel containing ethidium bromide (0.5 μg / ml). Under the ultraviolet...

Embodiment 2

[0103] 1. Stem cell targeting of targeting plasmid vector containing recombinant Krt9 gene

[0104] The targeting plasmid containing the recombinant Krt9 gene was treated with Not I to linearize it, and the DNA concentration was adjusted to about 1 μg / μl. Schematic diagram of mouse stem cell targeting operation process Figure 15 shown.

[0105] 2. Preparation of mouse stem cell feeder cells

[0106] 1) Production and cryopreservation of mouse embryonic fibroblasts (MEF cells):

[0107] ① C57BL6J female mice with a gestational age of 12.5d to 14.5d were killed by dislocation of the neck. After disinfection with 75% alcohol, a small incision was made in the abdominal cavity, and the abdominal cavity was carefully torn open to expose the abdominal cavity;

[0108] ② Change a set of scissors and tweezers, open the abdominal cavity, take out the embryo, put it into a 10cm Petri dish with 20ml PBS, and wash the blood;

[0109] ③Wash the embryo once with PBS and transfer to anot...

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Abstract

The invention discloses a method for constructing a human epidermolytic palmoplantar keratoderma mouse model. The method comprises the following steps: mutating A of the 434th nucleotide of mouse Krt9 genes into GGCT, constructing a targeting plasmid vector containing recombinant Krt9 genes shown as SEQ ID NO:1, and screening a mouse carrying the recombinant Krt9 genes, thereby obtaining the human epidermolytic palmoplantar keratoderma mouse model. The mouse is obtained by utilizing a gene targeting technology, an international first mouse strain of Krt9 gene knock-in mutation is screened, namely a Krt9-indel mouse, and the phenotype of the model is completely consistent with that of the human EPPK, so that a specific animal model of completely imitating the human EPPK from genes to the phenotype is obtained, and the model can be further widely applied to research of pathogenic causes and pathogenic mechanisms of human EPPK and screening of medicines for treating the EPPK and gene treatment tests.

Description

(1) Technical field [0001] The invention relates to a method for constructing a specific site c.434delAinsGGCT mutation animal model of Krt9 gene and the application of the animal model in human epidermolysis palmoplantar keratoderma. (2) Background technology [0002] Mice (Mus musculus) are a very good animal model that can be used to study the etiology, molecular mechanism, drug and treatment of human diseases. The gene targeting embryonic stem cell method is to partially replace the DNA sequence of the mouse target gene through homologous recombination, and the phenotype of the resulting mouse is a reflection of the important function of this part of the DNA sequence. Animal models obtained by gene targeting technology can be used to observe experimental animals as a whole and infer the function of corresponding genes, so this technology has been recognized internationally. The three scientists who invented this technology, Capecchi, Smithies and Evans, won the Nobel Pri...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/89A01K67/027
Inventor 张咸宁陈晓玲吕雅素高翔赵静刘荣荣姜湖铃柯海萍徐晨明杜振方
Owner ZHEJIANG UNIV
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