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A kind of extraction method of Wolbachia (wolbachia) used for embryo injection transfection

An extraction method and embryo technology, which are applied in the field of Wolbachia extraction, can solve problems such as being unfavorable to mosquito strains and take a long time, and achieve the effects of saving time and energy, easy operation and strong controllability

Active Publication Date: 2011-12-14
GUANGZHOU WOLBAKI BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The second method takes too long, which is not conducive to the rapid establishment of Wolbachia (Wolbachia) transfected mosquito strains.

Method used

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  • A kind of extraction method of Wolbachia (wolbachia) used for embryo injection transfection
  • A kind of extraction method of Wolbachia (wolbachia) used for embryo injection transfection
  • A kind of extraction method of Wolbachia (wolbachia) used for embryo injection transfection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] 1. Acquisition of Wolbachia: Collect the ovaries of Aedes albopictus containing Wolbachia by dissecting them, and dissect multiple mosquitoes at one time to collect their ovaries. The ovaries collected at one time are very important. About 50mg, collected and placed in 1ml of SPG buffer. The ovarian tissue was crushed with a Dounce Tissue Grinder, and the ovarian tissue was ground 10 times by the Dounce Tissue Grinder, 0.5 min each time, and the ground tissue was washed with 0.5 ml of SPG buffer collect. The collected liquid was then centrifuged at 300 g for 5 minutes in a centrifuge to remove some tissue fragments. Transfer the supernatant to a new centrifuge tube and centrifuge again at 12000g for 10 minutes. The supernatant was removed, and the sediment in the centrifuge tube contained purified Wolbachia. After dissolving the sediment in the centrifuge tube with 50 microliters of SPG buffer solution, centrifuge again at 300g for 5 minutes to further remove the rem...

Embodiment 2

[0023] 1. Acquisition of Wolbachia: Collect the ovaries of Aedes albopictus containing Wolbachia by dissecting them, and dissect multiple Aedes albopictus at one time to collect their ovaries. The ovary of Aedes albopictus weighs about 50 mg and is collected in 1 ml of SPG buffer. The ovarian tissue was crushed with a Dounce Tissue Grinder, and the ovarian tissue was ground 10 times by the Dounce Tissue Grinder, 0.5 min each time, and the ground tissue was washed with 0.5 ml of SPG buffer collect. The collected liquid was then centrifuged at 100g in a centrifuge for 10 minutes to remove some tissue fragments. Transfer the supernatant to a new centrifuge tube and centrifuge again at 15000g for 5 minutes. The supernatant was removed, and the sediment in the centrifuge tube contained purified Wolbachia. After dissolving the sediment in the centrifuge tube with 50 microliters of SPG buffer solution, centrifuge again at 100g for 10 minutes to further remove the remaining tissue ...

Embodiment 3

[0027] 1. The acquisition of Wolbachia (Wolbachia) is as follows: the Culex mosquito (Cx.pipiens molestus) containing Wolbachia (Wolbachia) is collected by dissecting its ovaries, and a plurality of Culex mosquitoes are dissected at one time to collect its ovaries. Culex ovaries collected at one time weigh about 50 mg, and placed in 1 ml of SPG buffer after collection. The ovarian tissue was crushed with a Dounce Tissue Grinder, and the ovarian tissue was ground 10 times by the Dounce Tissue Grinder, 0.5 min each time, and the ground tissue was washed with 0.5 ml of SPG buffer collect. The collected liquid was then centrifuged at 1000g in a centrifuge for 1 minute to remove some tissue fragments. Transfer the supernatant to a new centrifuge tube and centrifuge again at 5000g for 20 minutes. The supernatant was removed, and the sediment in the centrifuge tube contained purified Wolbachia. After dissolving the sediment in the centrifuge tube with 50 microliters of SPG buffer ...

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Abstract

The invention discloses a method for extracting wolbachia used for embryonic injection transfection, which comprises: dissecting an insect having wolbachia to obtain the ovary of the insect, and placing the ovary in sucrose phosphate glutamate (SPG) buffer solution; and homogenizing the ovary, washing the homogenate with the SPG buffer solution, collecting the homogenate, placing the collected homogenate in a centrifuge to centrifuge the homogenate in amount of 100 to 1,000 grams for 1 to 10 minutes, collecting supernate, centrifuging the supernate in an amount of 5,000 to 15,000 grams for 5 to 20 minutes, removing supernate, washing the sediment with the SPG buffer solution to obtain suspension, centrifuging the suspension in an amount of 100 to 1,000 grams for 1 to 10 minutes, collecting the supernate which is pure wolbachia, wherein the pure wolbachia can be used for embryonic injection in mosquitoes. In the method disclosed by the invention, the source (ovary tissues of insects having the wolbachia) of the wolbachia is rich and can be obtained easily; and the whole method is simple and convenient in operation, has very high controllability, and can obtain lots of wolbachia forembryonic injection transfection in a give time very easily, thereby saving lots time and energy, improving working efficiency and contributing to quick establishment of a mosquito strain transfectedwith wolbachia.

Description

Technical field: [0001] The invention belongs to the field of biotechnology, in particular to a method for extracting Wolbachia used for embryo injection transfection. Background technique: [0002] Wolbachia is a symbiotic microorganism widely distributed in arthropods, and it may be the most abundant group of insect symbiotic microorganisms. It is distributed in Coleoptera, Diptera, Hemiptera, Homoptera, Hymenoptera, Lepidoptera and other insect species. It uses vertical transmission as its basic mode of transmission among host generations. It exists stably in the germ cells of the host, is transmitted to the offspring of the host through egg cells, and can regulate the reproductive activities of the host through various methods such as cytoplasmic incompatibility, feminization and andricide. These regulatory functions promote its widespread spread within the host population. [0003] Cytoplasmic incompatibility (CI) is the most common type of host reproductive behavior...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20A01N63/00A01P7/04C12R1/01
Inventor 奚志勇朱俭
Owner GUANGZHOU WOLBAKI BIOTECH CO LTD
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