Construction and application of a 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: 2022-07-26
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 a phosphorylated mutant sufu transgenic mouse model based on homologous recombination technology
  • Construction and application of a phosphorylated mutant sufu transgenic mouse model based on homologous recombination technology
  • Construction and application of a phosphorylated mutant sufu transgenic mouse model based on homologous recombination technology

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Experimental program
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Effect test

Embodiment 1

[0043] will SUFU WT / SUFU S342 / 346D / SUFU S342 / 346A The encoding gene of the double phosphorylation mutant and the wild-type SUFU encoding gene were transferred into the pBigT vector containing the tpA transcription termination sequence, and then the pBigT vector containing the tpA transcription termination sequence and the target gene was loaded into 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 is possible to identify whether a fragment containing the gene of interest has been inserted. The...

Embodiment 2

[0050] Isolation of primary mouse embryonic fibroblasts: Pregnant mice at 14.5 d of gestation were sacrificed by cervical dislocation. The uterus and fetal membranes were torn open to take out the embryos and placed in sterile 1×PBS.

[0051] Remove the tail, limbs, head and internal organs with sterile forceps (all bright red tissues should be removed), leaving only the back tissue of the trunk, and wash the blood stains with sterile 1×PBS.

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

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

[0054] Add 1-2ml of 0.25% trypsin-EDTA to the centrifuge tube and mix it with the tissue fragments by repeated pipetting with a dropper.

[0055] It was placed in a cell culture incubator for 20 minutes, and then repeatedly pipetted with a dr...

Embodiment 3

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

[0059] First, the primary MEF cells were seeded in a 10 cm P100 culture dish. When the cell density reached 90%, the previous culture medium was discarded, and the cells were washed twice with 1×PBS before adding ligand or drug stimulation. The whole stimulation process All experiments were performed in serum-free DMEM without dual antibodies.

[0060] The specific consumption is as follows: serum-free DMEM without dual antibodies: ShhN=9:1; serum-free DMEM without dual antibodies: DMSO=1000:1; serum-free DMEM without dual antibodies: SAG=2000:1.

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

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Abstract

The invention provides the construction and application of a phosphorylated mutant SUFU transgenic mouse model based on homologous recombination technology, including the following steps: constructing a targeting vector pROSA26PA containing the phosphorylated mutant SUFU gene; 129 / S6ES cells were transfected; ES cell clones with correct homologous recombination were selected by PCR amplification and sequencing; ES cells were injected into blastocysts of C57BL / 6 mice by microinjection technology, and mated with C57BL / 6 mice. Brown and black mice were produced; positive mice were screened by PCR for brown mice, mated with Ddx4-Cre mice, and heterozygous F1 generation mice were obtained by PCR screening; F2 generation mice were obtained by self-crossing, and screened Homozygous F2 generation mice were obtained, namely, the phosphorylated mutant SUFU transgenic mouse model. The invention can be used to study the two-way regulation mechanism of SUFU on the Hedgehog (Hh) signal pathway, and introduce it into Ptch + / ‑ The pathogenesis of Hh subtype medulloblastoma can be further studied in the spontaneous medulloblastoma mouse model, which provides a powerful tool for screening a new generation of Hh antagonists against medulloblastoma; and this model mouse can also be used for research Effects of Hh on sperm development.

Description

technical field [0001] The invention relates to a method for constructing a mutant mouse model by phosphorylation at a specific site of a 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 across 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 the most common cerebellar tumor in children, accounting for 20% of all childhood brain cancers. This malignant tumor originates from granular neuron precursor cells (Granule Neuron Precursor Cells, GNPCs) in the cerebellar cortex, or deep neural stem cells (Neural Stem Cells). Granular neuron precursor cells in the outer gra...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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