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A method for establishing a mammary gland bioreactor

A bioreactor and mammary gland technology, applied in the fields of biotechnology and tissue engineering, can solve the problems of difficult to improve the success rate of mammary gland bioreactors, large trauma, and high difficulty in surgery

Inactive Publication Date: 2011-12-14
SOUTHERN MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There is nothing wrong with the method described in this patent application for breast tissue transplantation, but it is difficult to operate and requires a lot of work when it is used to establish a breast bioreactor and obtain valuable bioactive proteins by recovering milk.
In particular, this method causes great trauma to animals, and it is difficult to improve the success rate of establishing a mammary gland bioreactor, and the technical method is complicated, the production cost is high, and it is difficult to popularize and apply, especially for large animals suitable for establishing a mammary gland bioreactor. so so
In addition, the patent application mentioned in the background technology the method of isolating mammary gland stem cells from mammary gland tissue to transplant and regenerate mammary glands, but did not involve the direct introduction of exogenous target genes into mammary gland stem cells in animals

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1: Establishment of a mouse tPA mammary gland bioreactor.

[0035] (1) Prepare the transfection solution. Recipe: WAP-tPA gene fragment solution 50μl, add bFGF, lif and EGF respectively at a concentration of 20ng / ml; liposome 1μl, in which WAP is the mammary gland-specific expression promoter, tPA is the target gene, and liposome is the promoter dye.

[0036] (2) Breast duct injection. 5-8 days after the pregnancy of mice was selected as the mammary duct injection time. For the convenience of operation, it can be appropriately anesthetized, such as intraperitoneal injection of 0.5-1ml chloral hydrate; during operation, it is fixed on the operating table, and a Pasteur glass straw with a diameter of about 0.25mm that has been processed and blunted at the tip is inserted into the In the mammary gland duct of the mouse, the transfection solution is injected into the mammary gland tissue through this duct, and the injection volume is 30μl-50μl at a time.

[0037...

Embodiment 2

[0038] Example 2: Establishment of rabbit human albumin mammary gland bioreactor.

[0039] (1) Prepare the transfection solution. 20 μg of β-casein-albumin particles were added to 5 μl of liposomes, and bFGF, lif and SCF were added to 100 μl of normal saline at a concentration of 10 ng / ml after 15 minutes.

[0040] (2) Breast duct injection. The mammary gland injection time of selected rabbits was 8-12 days after pregnancy and 1-3 days after weaning. When injecting, the transfection solution is injected into the mammary gland tissue and the duct of the mammary gland by using a small amount of multi-point injection method.

[0041] (3) Detect the expression of the target gene. Four days after the female rabbit gave birth, the milk was collected, and the content of lactoferrin in the milk was detected by chemiluminescence method or immunoprecipitation method to be 1 g / L.

Embodiment 3

[0042] Example 3: Establishment of a mammary gland bioreactor for epidermal growth factor (EGF).

[0043] (1) Prepare the transfection solution. 5-15 mg of BLG-hEGF plasmid was added to 200-350 μl of liposomes, and after 15-20 minutes, 10-100 ml of physiological saline containing 5 ng / ml of bFGF and lif was added.

[0044] (2) Breast duct injection. The mammary duct injection time of selected goats at 4 months of age and 40-60 days of gestation. Because the teat duct of goats is thick, it can be observed with the naked eye, and the injection can be performed with a veterinary syringe and needle, and the operation is very convenient. The animal does not need to be anesthetized during the procedure, and the goat can be properly immobilized for safety. The amount of transfection solution injected each time is about 20-30ml. According to the situation, an additional injection can be made on the second and third days after the first injection, and the amount of each injection is...

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Abstract

The invention relates to a method for establishing a mammary gland bioreactor. The method is to introduce a target gene into the mammary duct or mammary gland tissue of an animal through a transfection solution, so that the target gene is expressed in the mammary gland under the control of a milk protein-specific regulatory sequence, It is characterized in that stem cell supporting factors are added to the transfection solution; the transfection solution containing the target gene is introduced in the animal's sexual development period or sexual maturity period or early pregnancy or weaning period to transfect mammary gland stem cells in the animal body. The establishment method of the mammary gland bioreactor of the present invention not only has a high success rate (50%-80%), low difficulty in operation, short production cycle and low cost, but also can be used repeatedly during the reproductive age of animals, and has a high commercial value.

Description

technical field [0001] The invention relates to the fields of biotechnology and tissue engineering, and further relates to a method for preparing a mutant by using a transfection liquid to introduce a target gene, in particular to a method for using a transfection liquid to introduce a target gene to establish a mammary gland bioreactor. Background technique [0002] The mammary gland bioreactor utilizes the very strong protein synthesis ability of animal breasts to produce precious natural bioactive substances through bioengineering means. In layman's terms, it is to use animal mammary gland cells as a natural fermenter for cell engineering to produce rare medicinal proteins, so that the biologically active substances that exist in a very small amount under natural conditions but are very effective are continuously produced from animal milk. This biological high-tech has attracted the attention of the world for its safety, environmental protection, conformity to the laws of...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/85C12N5/10
Inventor 安靓李进
Owner SOUTHERN MEDICAL UNIVERSITY
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