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Construction method and application of PDGF-BB transgenic mouse spinal cord transaction model

A PDGF-BB, transgenic mouse technology, applied to other methods of inserting foreign genetic materials, genetic engineering, chemical instruments and methods, etc., can solve the problem of unclear specific effects and underlying mechanisms, etc.

Inactive Publication Date: 2018-07-31
KUNMING MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although PDGF-BB has been shown to protect injured neurons from death and promote glial cell mitosis, its specific role in CNS injury and its underlying mechanism are still unclear and few reports have been involved, especially for the spinal cord Especially after total transverse trauma (SCT)

Method used

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  • Construction method and application of PDGF-BB transgenic mouse spinal cord transaction model
  • Construction method and application of PDGF-BB transgenic mouse spinal cord transaction model
  • Construction method and application of PDGF-BB transgenic mouse spinal cord transaction model

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0087] Example 1 Construction of PDGF-BB high expression transgenic mouse model

[0088] The PDGF-BB overexpression transgenic mice were constructed by transgenic technology, and the positive transgenic mice were identified by PCR. The strains with the highest and lowest expression of PDGF-BB were screened out by RT-PCR and Western blot techniques, and a mouse model with stable overexpression was established.

[0089] 1) Construction of PDGF-BB overexpression transgenic fragments, the construction method includes the following experimental steps:

[0090] 1.1) PCR cloning amplifies the open reading frame (ORF) of the target gene PDGF-BB, and the amplification primer sequence is as follows:

[0091] The upstream primer is 5'CGCTCCTTTGATGATCTCC 3', the nucleotide sequence of which is shown in SEQ ID NO.1;

[0092] The downstream primer is 5'TTGGTGCGGTCTATGAGG 3', its nucleotide sequence is shown in SEQ ID NO.2, and the target fragment is 230bp;

[0093] 1.2) pcDNA3.1(+)-h-PDGF...

Embodiment 2

[0121] Example 2 Construction of PDGF-BB Low Expression Transgenic Mouse Model

[0122] Using transgenic technology to construct transgenic mice with low expression of PDGF-BB, positive transgenic mice were identified by PCR, and the strains with the highest and lowest expression of PDGF-BB were screened out by RT-PCR and Western blot technology, and a mouse model with stable low expression was established.

[0123] 1) Construct a transgenic fragment with low expression of PDGF-BB, and identify the efficiency of PDGF-BB expression inhibition in vitro, including the following experimental steps:

[0124] 1.1) Construction of CMV-EmGFP-siRNA-PDGF-BB low-expression transgene fragment. like Image 6 As shown, the silent expression vector CMV-EmGFP-siRNA-PDGF-BB was purchased from Invitrogen, and the software provided by the company's website was used to target the target gene PDGF-BB (PDGFB platelet-derived growth factor beta polypeptide, GeneID: NM_011057) The point is (CGGCTGC...

Embodiment 3

[0138] Example 3 Establishment of a Transgenic Mouse Spinal Cord Transection Model

[0139] Using the obtained transgenic mouse model, establish a mouse model of total transection injury of the spinal cord. The specific steps include: anesthetize the transgenic mouse, fix it in the prone position, prepare the skin, cut the back skin, fascia and muscle layer, and remove the lamina with a rongeur , to expose the T9-T11 segmental spinal cord layer by layer, transect at the T10 level, and use a string with a diameter of about 1 mm to pass through the injury plane to ensure that the spinal cord is completely transected. After the operation is successful, the tail and limbs of the mouse can be observed to twitch. After the operation, the bleeding was stopped and sutured layer by layer. Mice whose spinal cord was successfully transected at T10 level showed postoperative paralysis of both lower limbs, no spontaneous movement, disappearance of urination radiation, and disappearance of...

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Abstract

The invention discloses a construction method and application of a PDGF-BB transgenic mouse spinal cord transection model. The construction method comprises the following steps that a transgenic miceare constructed, wherein the transgenic mice comprise PDGF-BB whole-body overexpression and low-expression mice; one transgenic mouse is anesthetized and fixed in a prostrate posture, skin is prepared, the skin on the back, fascia and muscle layers are cut open, a vertebral plate is removed by a bone rongeur, spinal cords of T9-T11 sections are exposed layer by layer and horizontally and transversely cut off on the T10 section, and a thin rope with the diameter of 1 mm penetrates through an injury plane to guarantee spinal cord transection; after an operation is successful, twitch of the tailand limbs of the mouse are observed; after the operation, bleeding is stopped, and layer-by layer sewing is conducted. The two lower limbs of the mouse obtained after successful spinal cord T10 horizontal transection are paralyzed after the operation, the mouse cannot move autonomously, micturition irradiation disappears, and the sense of pain and the sense of warmth of the two lower limbs disappear. It is found that mouse spinal cord injury repair, scar hyperplasia and function recovery are influenced after PDGF-BB overexpression and expression intervention.

Description

technical field [0001] The invention belongs to the technical field of animal model construction, and in particular relates to a construction method and application of a PDGF-BB transgenic mouse spinal cord transection model. Background technique [0002] Spinal cord injury (SCI) can lead to severe dysfunction and disability. It is a common clinical disease and will bring a heavy burden to patients and the whole society. It is frustrating that despite the efforts of neuroscience researchers all over the world for more than a hundred years, the treatment effect of SCI is still far from satisfactory. It was not until 1958, when Liu and Chambers confirmed for the first time that the adult mammalian CNS still had plasticity after injury, that people refocused their attention on the regeneration and repair of the CNS (including the spinal cord) after injury. [0003] Many studies have shown that the spinal cord has plasticity. This plasticity is manifested in the destruction of ...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N15/89A61D7/00A01K67/027
CPCC12N15/8509A01K67/0275A01K2207/05A01K2207/30A01K2227/105A01K2267/03A61D7/00C07K14/49C12N15/89C12N2800/107
Inventor 习杨彦彬胡滔陈波常业飞张丽王廷华张连峰
Owner KUNMING MEDICAL UNIVERSITY
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