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Application and method utilizing bombyx mori linnaeus vitellogenin promoter for gender-specific and tissue-specific expression of exogenous gene

A vitellogenin and promoter technology, applied in the direction of using vectors to introduce foreign genetic material, recombinant DNA technology, cells modified by introducing foreign genetic material, etc.

Active Publication Date: 2014-06-18
CAS CENT FOR EXCELLENCE IN MOLECULAR PLANT SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no related promoter reported in silkworm

Method used

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  • Application and method utilizing bombyx mori linnaeus vitellogenin promoter for gender-specific and tissue-specific expression of exogenous gene
  • Application and method utilizing bombyx mori linnaeus vitellogenin promoter for gender-specific and tissue-specific expression of exogenous gene
  • Application and method utilizing bombyx mori linnaeus vitellogenin promoter for gender-specific and tissue-specific expression of exogenous gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0096] Example 1. Construction of the carrier

[0097] The PXL-BACII-IE1-DSRED2 injection transfer sub-carrier used by the present invention was derived from Piggybac (see ELICK et al., GeNetica, 1996; WO2006122442), which was introduced in the Piggybac to seat carrier (Kojima, etc.Virusresearch, 2008) DSRED (NCBI login number: AJ851284; SEQIDNO: 2) driven by Virusresearch, constructs a PXL-BACII-IE1-DSRED2 rotor carrier.Family silkworm VGP promoter sequence comes from the 800bp sequence of the upstream of VG protein. It is obtained through segmented all-gene synthesis and cloned on the carrier PUC-57 (Sangonbiotech).-18T (TAKARA), build a plasmid PMD-18T-VGP. After the sequence is correct, use KPNI and APAI (NEB) to cut PMD-18T-VGP and PXL-BACII-IE1-DSRD2, and then insert VGP fragments into the carrierOn the plasmid PXL-BACII-IE1-DSRED2, the plasmid PXL-BACII-IE1-DSRED2-VGP is built.Sequenant on the 18T carrier to build a plasmid PMD-18T-EGFP. After obtaining the correct sequence...

Embodiment 2

[0098] Example 2. Test of BMN cell level

[0099] Transfer the three kinds of plasmids to the silkworm ovarian cells (BMN cells, see EP0225777), if the VGP promoter is activated and has organized specificity and gender specificity as predictive, that is, only in some genderExpression in specific organs, then green fluorescence will be observed in the experiment.The experimental cell transfection method refers to the EffectnetRansfectionReagentkit kit produced by QiaGen Company. The method is as follows: take 24 holes, select 12 holes, add 300UL BMN cells per hole, and cultivate overnight.The transfection particle PXL-BACII-IE1-DSRED2-EGFP, PXL-BACII-IE1-DSRD2-VGPEGFP, PXL-BACII-IE1-DSRED2-VGP are prepared, and the diluted concentration is 200ng / ul.The following operations are performed in the ultra -net platform: 70ul of EC is added to the EP tube of 1.5ml; then add 7.5ul of plasmid to add and mix; add 12ul of enhance, mix well, and settle 4 minutes; add 15ul of Efffectance, mix w...

Embodiment 3

[0102] Examples 3. Getting GMI silk silkworms and detection of specific expression of promoters

[0103] In this embodiment, three kinds of plasmids that only contain report genes, promoters and report genes, and only promoters, that is, PXL-BACII-IE1-DSRED2-EGFP, PXL-BACII-IE1-DSRED2-VGP-EGFP, PXL-BACII-IE1-DSRED2-VGP and PHA3pig (TAMURA et al., NatureBiotechnology, 2000, see EP1482035; used as an equivalent of the hybridase).In 4-8 hours), the purification kit of the injection plasmid DNA is purified by Qiagen's PlasmidMidikit kit. The injection method refers to the method described by Kanda & Tamura (1991) to perform micro-injection of silkworms.The injected silkworm eggs are seal with non -toxic glue to prevent pollution.In the conditions of 25 ° C, the green ant silkworms that are incubated and incubated will be performed.The expression of the promoter is detected.

[0104] The inventor detects the specific expression of the promoter through the following methods:

[0105]To ...

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Abstract

The invention provides an application and a method utilizing a bombyx mori linnaeus vitellogenin promoter for gender-specific and tissue-specific expression of an exogenous gene. The method breaks through a limitation that a traditional transgenic bombyx mori linnaeus cannot effectively perform male-specific, female-specific and tissue-specific expression of the exogenous gene, and develops and utilizes the bombyx mori linnaeus vitellogenin promoter for gender-specific and tissue-specific expression of the exogenous gene in bombyx mori linnaeus cell lines and the transgenic bombyx mori linnaeus for the first time. Therefore, with the first time of utilization of gender expression controllability and tissue expression controllability of the transgenic bombyx mori linnaeus, the application field of the bombyx mori linnaeus is expanded, the sustainable development of sericulture is maintained, and genetic control of insect pests can be studied.

Description

Technical field [0001] The invention involves biotechnology.Specifically, the present invention involves the use of Bombyxmorilinnaeus (Vitellogenin) promoter for gender specificity and tissue -specific expression of exogenous genes. Background technique [0002] Family silkworms are silk insects, and they are also important economic insects and model creatures among the bulk -winged insects. They have made huge contributions to my country's economic development and are further contributing to China's sustainable economic development.With the completion of our national silkworm genome plan to announce the completion of the framework of the silkworm genome, marking the arrival of the genome era after the family silkworm, the main tasks of functional genome include: clone identification of a large number of functional genes, molecular regulation mechanisms for important biological traits, and realization of human beings.Control and regulate these genes and traits to meet the needs ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/63C12N5/10C12N15/85A01K67/033A01K67/04
Inventor 谭安江许军尤朗黄勇平
Owner CAS CENT FOR EXCELLENCE IN MOLECULAR PLANT SCI