Preparation method for transgenic mice capable of producing human nerve growth factor

A technology of nerve growth factor and transgenic mice, which is applied in the fields of molecular genetics and cell biology, can solve the problem of incorrect deletion of endogenous NGF gene in mice, achieve high success rate, simple operation, and overcome expression level unstable effect

Active Publication Date: 2015-04-29
深圳市国创纳米抗体技术有限公司
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  • Abstract
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  • Application Information

AI Technical Summary

Problems solved by technology

However, there are many difficulties or risks in obtaining mice with NGF gene knock-in by conventional gene knock-in technology. Screening, a negative screening to obtain positive clones
After the homolo...

Method used

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  • Preparation method for transgenic mice capable of producing human nerve growth factor
  • Preparation method for transgenic mice capable of producing human nerve growth factor
  • Preparation method for transgenic mice capable of producing human nerve growth factor

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Experimental program
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Effect test

Embodiment 1

[0041] 1. Construction of homologous recombination template vector

[0042] The homologous recombination template vector was constructed based on the pBR322 plasmid, and its structure was as follows: figure 1 shown. The constructed vector contains 5'-homologous recombination arm, mouse NGF gene third intron sequence, FRT, puromycin resistance gene (Puro gene), FRT, mouse NGF signal peptide sequence, human NGF gene, Mouse NGF 3'-untranslated region sequence, 3'-homologous recombination arm. Through homologous recombination, the human NGF gene can be replaced in situ by the mouse NGF gene, and at the same time, a puromycin resistance gene is introduced upstream at the 5'-end, and there is an FRT site at each of its two ends, which can be passed through the FLP recombinase Effect, removal of the puromycin resistance gene. However, the upstream and downstream DNA sequences of mouse NGF did not change.

[0043] The sequence of the 5'-homologous recombination arm can be shown in...

Embodiment 2

[0092] Expression detection of human NGF in the submandibular gland of knock-in mice

[0093] 1) Human NGF gene knock-in homozygous mice and wild-type mice were sacrificed by neck dislocation, the submandibular glands were taken and frozen sections were 4-8 μm in size, left at room temperature for 30 minutes, fixed in acetone at 4°C for 10 minutes, washed with PBS, 5 minutes × 3 times. Incubate with 3% hydrogen peroxide for 5-10 minutes to eliminate the activity of endogenous peroxidase. Wash with PBS, 5 minutes x 2 times.

[0094] 2) Block with 5-10% normal goat serum (diluted in PBS), and incubate at room temperature for 10 minutes. Pour off the serum, do not wash, add rabbit anti-human NGF antibody diluted in an appropriate proportion dropwise, and incubate at 37°C for 1-2 hours or overnight at 4°C. Rinse with PBS, 5 minutes x 3 times.

[0095] 3) Add fluorescein-labeled goat anti-rabbit secondary antibody (diluted in 1% BSA-PBS) in an appropriate proportion dropwise, i...

Embodiment 3

[0097] Embodiment 3: the acquisition and purification of NGF

[0098] 1. The mouse submandibular glands stored at -20°C were thawed in 0.5mM EDTA solution and homogenized (2ml / g tissue), then centrifuged at 9000 rpm at 4°C for 30 minutes, and the supernatant was taken and freeze-dried. The sample was redissolved with 4 ml of 50 mM PBS solution (pH 6.8), and dialyzed against the same buffer for 72 hours. The medium was changed 3 times in the middle. Then the CM-52 ion exchange resin (25 * 40 centimeters) that dialysate is balanced on the sample after dialysis, collects the effluent of OD. Then dialyze with 20 times the volume of 25mM PBS solution (pH6.8) for 72 hours, and change the solution three times.

[0099] 2. Replace the dialysate with 50mM sodium acetate / sodium chloride buffer (pH4.0), continue dialysis for 72 hours, and collect the supernatant after centrifugation.

[0100] 3. The CM-52 resin column (5×25cm) equilibrated with 50mM sodium acetate / sodium chlorid...

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Abstract

The invention discloses a method for producing transgenic mice through a homologous recombination technology. The method comprises the following steps: replacing mouse NGF genes on mouse chromosomes by human NGF genes through a Cas-9/CRISPR gene knock-in technology, and obtaining the genes with homozygous human NGF genes through breeding and knocking the genes in mice, thus obtaining filial generation mice with salivary glands capable of secreting human NGF. Compared with the conventional targeting technology utilizing positive and negative double screening homologous recombination genes, the method disclosed by the invention is simple to operate, capable of being realized only by transfecting mouse embryonic stem cells by three plasmid DNAs or carrying out pronucleus injection on the zygotes of mice, and high in success rate which is up to 2-5% and remarkably higher than the mouse embryonic stem cell positive rate of 0.1% of the common gene targeting technology.

Description

technical field [0001] The invention relates to a method for producing transgenic mice and further producing human nerve growth factor, belonging to the fields of molecular genetics and cell biology. Background technique [0002] Nerve growth factor (NGF) is the earliest neurotrophic factor discovered and the most thoroughly studied at present. It is a kind of nerve cell growth regulator with dual biological functions of neuron nutrition and spur growth. Development, differentiation, growth, regeneration, and expression of functional properties are all important regulators. NGF contains three subunits of α, β, and γ, and the active region is the β subunit, which is a dimer composed of two single chains of 118 amino acids bound by non-covalent bonds. At present, most of the clinically used NGF is the product of animal (mouse) gene expression. Although the biological effect of NGF has no obvious interspecies specificity, studies have shown that human NGF has significantly hig...

Claims

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Application Information

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IPC IPC(8): C12N15/85A01K67/027C07K14/48
Inventor 宋海鹏李敏王庆东
Owner 深圳市国创纳米抗体技术有限公司
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