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Construction and application of phosphorylated mutant SUFU transgenic mouse model based on homologous recombination technology

A technology of transgenic mice and homologous recombination, which is applied in the field of basic medicine, can solve problems such as the molecular mechanism is not very clear, and achieve the effect of good positive regulation

Active Publication Date: 2021-12-24
NANJING MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the exact molecular mechanism is still unclear

Method used

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  • Construction and application of phosphorylated mutant SUFU transgenic mouse model based on homologous recombination technology
  • Construction and application of phosphorylated mutant SUFU transgenic mouse model based on homologous recombination technology
  • Construction and application of phosphorylated mutant SUFU transgenic mouse model based on homologous recombination technology

Examples

Experimental program
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Effect test

Embodiment 1

[0043] will SUFU WT / SUFU S342 / 346D / SUFU S342 / 346A The double phosphorylation mutant coding gene and the wild-type SUFU coding gene are reprinted on the pBigT vector containing the tpA transcription termination sequence, and then the pBigT vector containing the tpA transcription termination sequence and the target gene is loaded onto the pRosa26PA containing the genomic DNA Rosa26 sequence, The pBigT sequence was integrated into the mouse genome by homologous recombination.

[0044] A forward primer Rosa26-F8731 (5'-CTTGCTCTCCCAAAGTCGCT-3') was designed on the Rosa26 sequence upstream of pBigT;

[0045] A reverse primer pBigT-R (5'-CGGCCTCGACTCTACGATAC-3') was designed on pBigT;

[0046] Another reverse primer Rosa26-R (5'-GCGGGAGAAATGGATATGAAGTA-3') was designed on the Rosa26 sequence downstream of pBigT.

[0047] With the help of these two pairs of primers, it can be identified whether a fragment containing the gene of interest is inserted. The identification criteria...

Embodiment 2

[0050] Isolation of primary mouse embryonic fibroblasts: Pregnant mice at 14.5 days of pregnancy were killed by neck dislocation, and the uterus and fetal membranes were routinely torn apart to take out the embryos and place them in sterile 1×PBS.

[0051] Use sterile tweezers to remove the tail, limbs, head and viscera (all bright red tissues should be removed), leaving only the back tissues of the trunk, and wash the blood stains with sterile 1×PBS.

[0052] Use a sterile scalpel to cut the back tissue of the trunk into 1-3mm pieces as much as possible.

[0053] Transfer the pieces of the same embryo to the same 15ml centrifuge tube, let it stand until the tissue pieces sink to the bottom of the tube, and carefully aspirate and discard the supernatant.

[0054] Add 1-2ml 0.25% trypsin-EDTA to the centrifuge tube, and repeatedly pipette with a dropper to mix it with the tissue fragments evenly.

[0055] Place it in a cell culture incubator and let it stand for 20 minutes, an...

Embodiment 3

[0058] Add ShhN ligand or Smoothen's agonist SAG to primary MEF cells to simulate the activation state of Hedgehog signaling pathway in MEF cells:

[0059] First, the primary MEF cells were planted in a 10cm P100 culture dish. When the cell density reached 90%, the previous culture medium was discarded, and the cells were washed twice with 1×PBS, ready to be stimulated with ligand or drug. The whole stimulation process All were carried out in DMEM without serum and double antibody.

[0060] The specific dosage is as follows: serum-free double-antibody-free DMEM: ShhN=9:1; serum-free double-antibody-free DMEM: DMSO=1000:1; serum-free double-antibody-free DMEM: SAG=2000:1.

[0061] After 24 hours of stimulation, the total RNA and total protein of the cells can be collected for subsequent experiments.

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Abstract

The invention provides a construction and application of a phosphorylated mutant SUFU transgenic mouse model based on a homologous recombination technology. The construction comprises the following steps that a targeting vector pROSA26PA containing a phosphorylated mutant SUFU gene is constructed; linearization is carried out, and then 129 / S6ES cells are transfected; amplifying and sequencing by using a PCR method are achieved to screen ES cell clones with correct homologous recombination; ES cells are injected into blastocysts of C57BL / 6 mice through a microinjection technology and mated with the C57BL / 6 mice, and brown and black mice are generated; positive mice is screened out from the brown mice by using the PCR method, the positive mice are mated with Ddx4-Cre mice, and PCR screening is carried out to obtain hybrid F1-generation mice; and selfing is achieved to obtain F2-generation mice, and homozygous F2-generation mice are screened out, namely the phosphorylated mutant SUFU transgenic mouse model. The construction and application of the phosphorylated mutant SUFU transgenic mouse model based on the homologous recombination technology can be used for researching a bidirectional regulation mechanism of SUFU on a Hedgehog (Hh) signal channel, the pathogenesis of Hh subtype medullary cell carcinoma can be further researched by introducing the SUFU into a Ptch + / -spontaneous medullary cell carcinoma mouse model, and a powerful tool is provided for screening a new generation of Hh antagonist for resisting the medullary cell carcinoma; and a model mouse can also be applied to research on the influence of Hh on sperm development.

Description

technical field [0001] The invention relates to a method for constructing a specific site phosphorylation modification mutant mouse model of human gene SUFU and the application of the mouse model in the study of medulloblastoma carcinogenesis and sperm development, belonging to the technical field of basic medicine. Background technique [0002] The Hedgehog (Hh) signaling pathway is conserved in various species from insects to humans, and its main role is to regulate embryonic development and adult tissue homeostasis. Abnormal function of this signal can lead to the occurrence of medulloblastoma (MB) and affect sperm development. [0003] Medulloblastoma is a common cerebellar tumor in children, accounting for 20% of all childhood brain cancers. This malignant tumor originates from the granular neuron precursor cells (Granule Neuron Precursor Cells, GNPCs) in the cerebellar cortex, or the deep neural stem cells (Neural Stem Cells). Granular neuron precursor cells in the o...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/12C12N15/85C12N15/877A01K67/027
CPCC12N5/0606C12N15/8509C12N15/8775C07K14/47A01K67/0278A01K2207/15A01K2217/072A01K2227/105A01K2267/0331
Inventor 程雁刘晨韩博昂王瑜张子宇乐珅俞婷婷
Owner NANJING MEDICAL UNIV
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