CN104404072A - Method for predicting expression quantity of exogenous gene and screening transgenic organism with marker gene - Google Patents
Method for predicting expression quantity of exogenous gene and screening transgenic organism with marker gene Download PDFInfo
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- CN104404072A CN104404072A CN201410678673.2A CN201410678673A CN104404072A CN 104404072 A CN104404072 A CN 104404072A CN 201410678673 A CN201410678673 A CN 201410678673A CN 104404072 A CN104404072 A CN 104404072A
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Abstract
The invention discloses a method for predicting the expression quantity of an exogenous gene and screening a transgenic organism with a marker gene. A transgenic expression plasmid comprising two expression cassettes of the marker gene and the exogenous gene is built by adopting a molecular biological technique, wherein the two expression cassettes are adjacent and the same in transcription direction. Microinjection is used for leading the transgenic expression plasmid and an auxiliary plasmid into a genome of an organism together and then the transgenic organism whose exogenous gene can achieve stable heredity and expression is cultured; the expression quantity of the exogenous gene can be predicted based on information of the expression quantity of the marker gene in the transgenic organism, and a biological organism or variety corresponding to the expression quantity of the exogenous gene is obtained through screening. The method can be used for screening the transgenic organism or variety capable of expressing the exogenous gene efficiently from a large quantity of transgenic organisms easily, quickly and conveniently, thereby providing an ideal screening means for development of an efficient biological reactor.
Description
Technical field
The method that the present invention relates to a kind of prediction in genetically modified organism field and screen, especially relates to the method for a kind of marker gene prediction exogenous gene expression amount and screening transgenic biology.
Background technology
Utilize Protocols in Molecular Biology to be inserted in organism genome by foreign gene, enable foreign gene genetic stability and expression, this technology is called transgenic technology, and corresponding biology is called genetically modified organism.If the expression product of foreign gene is protein, and be a closed system, as expressed in the systems such as mammary gland, bladder, sericterium, this genetically modified organism is called bio-reactor.
In most transgenic research, foreign gene is all generally random be inserted in host genome, is distributed in the regions such as intergenic region, gene intron, gene extron.Its expression amount of foreign gene of different insertion point can be variant, and this phenomenon is called as position effect.Therefore, want the genetically modified organism screening, obtain foreign gene great expression, just must carry out qRT-PCR, Western equimolecular biological assay to a large amount of trans genie individual, to determine excellent individual, the workload of screening is large, length consuming time, this has become the bottleneck of development and utilization genetically modified organism.
Summary of the invention
The object of this invention is to provide the method for a kind of marker gene prediction exogenous gene expression amount and screening transgenic biology, utilize two tight adjacent, that expression cassette exogenous gene expression measurer in the same way has significant correlation features, according to marker gene expression amount prediction exogenous gene expression amount, also screening transgenic is biological further, effectively simple, this can filter out trans genie individual or the kind of efficiently expressing exogenous gene rapidly from a large amount of trans genie individual, lays the foundation for accelerating to build efficient bio-reactor.
In order to achieve the above object, the step of the technical solution used in the present invention is as follows:
1) adopt Protocols in Molecular Biology to build the transgene expression plasmid including marker gene and foreign gene two expression cassettes, the adjacent spaces of marker gene and foreign gene two expression cassettes is less than 30bp and transcriptional orientation is identical;
2) adopt the transgenosis plasmid that step 1) obtained of microinjection and transposon in for transgenosis plasmid to provide together with the helper plasmid of transposase to import in biological genome, cultivating into foreign gene can the genetically modified organism of genetic stability and expression;
3) the marker gene expression amount in the genetically modified organism obtained and exogenous gene expression measurer have significant correlation, carry out predicting and screening according to the information of genetically modified organism marker gene expression amount, prediction exogenous gene expression amount, and screening obtains biont corresponding to this exogenous gene expression amount or kind.
Preferably, the succession of described transgene expression plasmid marker gene and foreign gene two expression cassettes is any.
Preferably, described marker gene adopts EGFP marker gene.
Preferably, described foreign gene adopts firefly luciferase gene (Fluc) or renilla luciferase (Rluc) gene.
Preferably, described host adopts silkworm.
Preferably, described transgenosis plasmid and the blending ratio of helper plasmid are 1:0.5 ~ 1:1.
The beneficial effect that the present invention has is:
The present invention utilizes two tight adjacent, that expression cassette exogenous gene expression measurer in the same way has significant correlation features, and the expression level of serviceable indicia gene predicts foreign gene expression levels adjacent with it.This method is compared traditional genetically modified organism foreign gene expression levels and is detected, and has advantage that is easy, that save time, save money.Because the detection gimmick of marker gene is many, technology maturation, so it can filter out the genetically modified organism of efficiently expressing exogenous gene fast and efficiently from a large amount of trans genie individual, improving genetically modified organism screening efficiency, providing desirable screening means for setting up efficient bio-reactor.
Embodiment
Below in conjunction with embodiment, the invention will be further described.
Embodiments of the invention are as follows:
Embodiment 1:
The present embodiment constructs a foreign gene in front, marker gene rear, and with
piggyBactransgenosis plasmid pBSer1Fluc-A3EGFP based on transposon, in transgenosis plasmid, the expression cassette of Photinus pyralis LUC (Fluc) foreign gene is started by glue protein 1 gene (Ser1) promotor that silkworm middle division of silkgland is specific expressed, the expression cassette of green fluorescent protein (EGFP) marker gene is started by Actin muscle A3 promotor, sericin 1 gene (Ser1) promotor that wherein silkworm middle division of silkgland is specific expressed has intercepted the proximal promoter of 575bp length, the adjacent spaces of Photinus pyralis LUC (Fluc) exogenous gene expression frame and green fluorescent protein (EGFP) marker gene expression cassette is 25bp.
By pBSer1Fluc-A3EGFP transgenosis plasmid be
piggyBactransposon provides the helper plasmid of transposase to be dissolved in pH7.6 phosphoric acid buffer together, and in phosphoric acid buffer, pBSer1Fluc-A3EGFP plasmid and helper plasmid press the mixing of 1:0.5 ratio, and the injection total concn of two kinds of plasmids is 0.4 μ g/ μ l.
Then, in the zygote adopting microinjection to be laid eggs in latter 8 hours by mixing plasmid importing silkworm, injection cumulative volume is about 10nl.Silkworm seed after injection is raised to adult under the condition of 25 DEG C of temperature, 85% humidity, 12h illumination, and selfing obtains G1 generation.At G1 for little silkworm rearing season, utilize Japanese Olympus SZX12 fluorescent microscope, filter out the positive silkworm of expressing EGFP marker gene, the excitation wavelength of fluorescent microscope is 460nm ~ 490nm, and emission wavelength is 510 nm ~ 550 nm.When G1 raises to the 3rd day five ages for transgenic bombyx mori, get 7 transgenic bombyx mori moth districts altogether, due to
piggyBactransposon inserts genomic randomness, and 7 identified proofs in transgenic bombyx mori moth district have different insertion point, and the middle division of silkgland that 3 silkworms are got in each moth district repeats as 3 biology.
Adopt fluorescent quantitative PCR technique, be that reference gene detects foreign gene Fluc in 2 order arrangement expression cassettes and the transcriptional level of marker gene EGFP with Rp49, correlation statistics analytical results shows that the correlationship of Fluc and the EGFP transcriptional level of 7 transgenic bombyx mori kinds reaches pole conspicuous level, and concrete outcome is: coefficient of determination R
2=0.8382, correlation coefficient r=0.9155, probable value P < 0.01.In this digital proof genetically modified organism, 2 tight adjacent, expression cassette exogenous gene expression measurers in the same way have significant correlation, even if foreign gene insertion point is different, because marker gene is closely adjacent with foreign gene two expression cassettes, so still there is pole significant correlation with the transcriptional level of marker gene EGFP in the transcriptional level of foreign gene Fluc.
Embodiment finally detects according to EGFP fluorescence power transgenic silkworm, judges EGFP marker gene expression amount with this, and prediction Fluc exogenous gene expression amount, it is individual that screening obtains silkworm corresponding to Fluc exogenous gene expression amount.
Embodiment further to 8 G2 of another identical transgenic experiments for transgenic lines, detect according to EGFP fluorescence power, judged EGFP marker gene expression amount with this, predict Fluc exogenous gene expression amount.First determining the highest kind of marker gene EGFP transcriptional level is ZY153, experiment uses 8 G2 individual for other of transgenic lines again, actually have detected Fluc marker gene expression level, results contrast proves that the transcriptional level of the foreign gene Fluc of this kind is the highest really, even if the Rp49 adopting high level expression is reference gene, its relative transcript levels still reaches 3.3%.Embodiment proves to utilize marker gene EGFP expression amount to predict foreign gene Fluc expression amount, and screening obtains the highest transgenic bombyx mori kind of foreign gene Fluc expression amount further.
Although there is the process of a post transcriptional modificaiton from mRNA to protein, but because the post transcriptional modificaiton process of same gene is the same, so for same gene, have selected the kind that transcriptional level is the highest, namely have selected the highest kind of protein expression level.
Embodiment 2:
The present embodiment constructs a foreign gene in front, marker gene rear, and with
piggyBactransgenosis plasmid pBSer1Fluc-A3EGFP based on transposon, in transgenosis plasmid, the expression cassette of Photinus pyralis LUC (Fluc) foreign gene is started by glue protein 1 gene (Ser1) promotor that silkworm middle division of silkgland is specific expressed, the expression cassette of green fluorescent protein (EGFP) marker gene is started by Actin muscle A3 promotor, sericin 1 gene (Ser1) promotor that wherein silkworm middle division of silkgland is specific expressed has intercepted the proximal promoter of 4000bp length, and the adjacent spaces of exogenous gene expression frame and marker gene expression cassette is 25bp.
By pBSer1Fluc-A3EGFP transgenosis plasmid be
piggyBactransposon provides the helper plasmid of transposase to be dissolved in pH7.6 phosphoric acid buffer together, and in phosphoric acid buffer, pBSer1Fluc-A3EGFP plasmid and helper plasmid press the mixing of 1:0.6 ratio, and the injection total concn of two kinds of plasmids is 0.4 μ g/ μ l.
Then, in the zygote adopting microinjection to be laid eggs in latter 8 hours by mixing plasmid importing silkworm, injection cumulative volume is about 10nl.Silkworm seed after injection 25 DEG C, 85% humidity, 12h illumination condition under raise to adult, selfing obtains G1 generation.At G1 for little silkworm rearing season, utilize Japanese Olympus SZX12 fluorescent microscope, filter out the positive silkworm of expressing EGFP marker gene, the excitation wavelength of fluorescent microscope is 460nm ~ 490nm, and emission wavelength is 510 nm ~ 550 nm.When G1 raises to the 3rd day five ages for transgenic bombyx mori, get 5 transgenic bombyx mori moth districts altogether, due to
piggyBactransposon inserts genomic randomness, and 5 identified confirmations in transgenic bombyx mori moth district have different insertion point, and the middle division of silkgland that 3 silkworms are got in each moth district repeats as 3 biology.Adopt fluorescent quantitative PCR technique, be that reference gene detects foreign gene Fluc in 2 order arrangement expression cassettes and the transcriptional level of marker gene EGFP with Rp49, correlation statistics analytical results shows that the correlationship of Fluc and the EGFP transcriptional level of 5 transgenic bombyx mori kinds reaches pole conspicuous level, and concrete outcome is: coefficient of determination R
2=0.9871, correlation coefficient r=0.9935, significance value P < 0.01.This data consistent with Example 1 is identical, to again demonstrate in genetically modified organism 2 tight adjacent, expression cassette exogenous gene expression measurers in the same way and have significant correlation, even if foreign gene insertion point is different, because marker gene is closely adjacent with foreign gene two expression cassettes, so still there is pole significant correlation with the transcriptional level of marker gene EGFP in the transcriptional level of foreign gene Fluc.
Embodiment finally detects according to EGFP fluorescence power transgenic silkworm, judges EGFP marker gene expression amount with this, and prediction Fluc exogenous gene expression amount, it is individual that screening obtains silkworm corresponding to Fluc exogenous gene expression amount.
Embodiment further to 6 G2 of another identical transgenic experiments for transgenic lines, detect according to EGFP fluorescence power, judged EGFP marker gene expression amount with this, predict Fluc exogenous gene expression amount.First determining the highest kind of marker gene EGFP transcriptional level is ZY148, experiment uses 6 G2 individual for other of transgenic lines again, actually have detected Fluc marker gene expression level, results contrast proves that the transcriptional level of the foreign gene Fluc of this kind is the highest really, take Rp49 as reference gene, its relative transcript levels reaches 2.1%.Embodiment proves to utilize marker gene EGFP expression amount to predict foreign gene Fluc expression amount, and screening obtains the highest transgenic bombyx mori kind of foreign gene Fluc expression amount further.
The principle of the present embodiment is with embodiment 1.
Embodiment 3:
The present embodiment constructs a foreign gene in rear, marker gene front, and with
piggyBactransgenosis plasmid pBA3EGFP-Fib-L-Rluc based on transposon, in transgenosis plasmid, the expression cassette of renilla luciferase (Rluc) foreign gene is started by silk fibroin light chain (Fib-L) promotor that Bombyx mori posterior silkgland is specific expressed, the expression cassette of green fluorescent protein (EGFP) marker gene is started by Actin muscle A3 promotor, silk fibroin light chain (Fib-L) promotor that wherein Bombyx mori posterior silkgland is specific expressed has intercepted the proximal promoter of 690bp length, and the adjacent spaces of exogenous gene expression frame and marker gene expression cassette is 30bp.
By pBA3EGFP-Fib-L-Rluc transgenosis plasmid be
piggyBactransposon provides the helper plasmid of transposase to be dissolved in pH7.6 phosphoric acid buffer together, and in phosphoric acid buffer, pBA3EGFP-Fib-L-Rluc plasmid and helper plasmid press the mixing of 1:1 ratio, and the injection total concn of two kinds of plasmids is 0.4 μ g/ μ l.
Then, in the zygote adopting microinjection to be laid eggs in latter 8 hours by mixing plasmid importing silkworm, injection cumulative volume is about 10nl.Silkworm seed after injection 25 DEG C, 85% humidity, 12h illumination condition under raise to adult, selfing obtains G1 generation.At G1 for little silkworm rearing season, utilize Japanese Olympus SZX12 fluorescent microscope, filter out the positive silkworm of expressing EGFP marker gene, the excitation wavelength of fluorescent microscope is 460nm ~ 490nm, and emission wavelength is 510 nm ~ 550 nm.When G1 raises to the 3rd day five ages for transgenic bombyx mori, get 6 transgenic bombyx mori moth districts altogether, due to
piggyBactransposon inserts genomic randomness, 6 identified proofs in transgenic bombyx mori moth district have different insertion point, the posterior division of silkgland that 3 silkworms are got in each moth district repeats as 3 biology, adopt fluorescent quantitative PCR technique, be that reference gene detects foreign gene Rluc in 2 order arrangement expression cassettes and the transcriptional level of marker gene EGFP with GAPDH, correlation statistics analytical results shows that the correlationship of Rluc and the EGFP transcriptional level of 6 transgenic bombyx mori kinds reaches pole conspicuous level, and concrete outcome is: concrete outcome is R
2=0.8161, r=0.9034, P < 0.01.These data prove further, even if foreign gene makes Rluc into, but 2 tight adjacent, expression cassette exogenous gene expression measurers in the same way have this characteristic of significant correlation not change in genetically modified organism, even if foreign gene insertion point is different, because marker gene is closely adjacent with foreign gene two expression cassettes, so still there is pole significant correlation with the transcriptional level of marker gene EGFP in the transcriptional level of foreign gene Rluc.
Embodiment finally detects according to EGFP fluorescence power transgenic silkworm, judges EGFP marker gene expression amount with this, and prediction Fluc exogenous gene expression amount, it is individual that screening obtains silkworm corresponding to Fluc exogenous gene expression amount.
Embodiment further to 9 G2 of another identical transgenic experiments for transgenic lines, detect according to EGFP fluorescence power, judged EGFP marker gene expression amount with this, predict Rluc exogenous gene expression amount.First determining the highest kind of marker gene EGFP transcriptional level is ZWB8, experiment uses 9 G2 individual for other of transgenic lines again, actually have detected Rluc marker gene expression level, results contrast proves that the transcriptional level of the foreign gene Rluc of this kind is the highest really, is that its relative transcript levels of reference gene reaches 5% with GAPDH.Embodiment proves to utilize marker gene EGFP expression amount to predict foreign gene Fluc expression amount, and screening obtains the highest transgenic bombyx mori kind of foreign gene Rluc expression amount further.
The principle of the present embodiment is with embodiment 1.
In sum, even if it is different that result of the present invention demonstrates foreign gene insertion point, under the condition that marker gene is closely adjacent with foreign gene two expression cassettes, there is pole significant correlation in transcriptional level and the transcribing of marker gene EGFP of foreign gene Fluc or Rluc.Therefore, marker gene can be utilized to predict the transcriptional level of foreign gene, thus simplify the program of screening efficiently expressing exogenous gene kind from a large amount of transformed variety, there is advantage that is easy, that save time, save money, providing desirable screening means for setting up efficient bio-reactor.
In all genetically modified organisms, gene expression pattern is identical, is all transcribed expression under promotor effect, has common point.No matter this experiment proves that foreign gene is Fluc gene, or Rluc gene, all there is pole significant correlation with the transcriptional level of marker gene EGFP in its transcriptional level.As long as can predict foreign gene and marker gene to be in state contiguous and in the same way, necessarily also there is pole significant correlation in both gene expression doses; And this dependency also necessarily exists when adopting other marker gene such as red fluorescent protein gene (DsRed), chloramphenicol acetyl transferasegene (cat), beta-glucosiduronatase gene (gus).
In embodiment
piggyBac(PB) transposon finds first in lepidopterous insects cabbage looper (Trichoplusia ni) clone TN-368.Afterwards, in the insects such as medfly (Ceratitis capitata), drosophila melanogaster (Dro-sophila melanogaster), Aedes aegypti (Aedes aegypti) and silkworm (Bombyx mori L.), PB transposon system is utilized all to achieve the success of transgenic experiments.Research also proves that PB transposon is the transposon system of a wide spectrum, at yeast (Schizosaccharomyces pombe), planaria (Girardia tigrina), zebra fish (Danio rerio), chicken, ox, pig, paddy rice and people's cell etc., successfully realize transgenosis from invertebrates to having the multiple biologies such as vertebrates.As can be seen here, although the species that embodiment adopts are silkworms, the present invention is applicable to other species too, has significant technique effect.
Above-mentioned embodiment is used for explaining and the present invention is described, instead of limits the invention, and in the protection domain of spirit of the present invention and claim, any amendment make the present invention and change, all fall into protection scope of the present invention.
Claims (6)
1. predict a method for exogenous gene expression amount and screening transgenic biology by marker gene, it is characterized in that the step of the method is as follows:
1) adopt Protocols in Molecular Biology to build the transgene expression plasmid including marker gene and foreign gene two expression cassettes, the adjacent spaces of marker gene and foreign gene two expression cassettes is less than 30bp and transcriptional orientation is identical;
2) adopt the transgenosis plasmid that step 1) obtained of microinjection and transposon in for transgenosis plasmid to provide together with the helper plasmid of transposase to import in biological genome, cultivating into foreign gene can the genetically modified organism of genetic stability and expression;
3) the marker gene expression amount in the genetically modified organism obtained and exogenous gene expression measurer have significant correlation, carry out predicting and screening according to the information of genetically modified organism marker gene expression amount, prediction exogenous gene expression amount, and screening obtains biont corresponding to this exogenous gene expression amount or kind.
2. the method for a kind of marker gene prediction exogenous gene expression amount according to claim 1 and screening transgenic biology, is characterized in that: the succession of described transgene expression plasmid marker gene and foreign gene two expression cassettes is any.
3. the method for a kind of marker gene prediction exogenous gene expression amount according to claim 1 and screening transgenic biology, is characterized in that: described marker gene adopts EGFP marker gene.
4. the method for a kind of marker gene prediction exogenous gene expression amount according to claim 1 and screening transgenic biology, is characterized in that: described foreign gene adopts firefly luciferase gene or renilla luciferase gene.
5. the method for a kind of marker gene prediction exogenous gene expression amount according to claim 1 and screening transgenic biology, is characterized in that: described host adopts silkworm.
6. the method for a kind of marker gene prediction exogenous gene expression amount according to claim 1 and screening transgenic biology, is characterized in that: described transgenosis plasmid and the blending ratio of helper plasmid are 1:0.5 ~ 1:1.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108728477A (en) * | 2017-04-24 | 2018-11-02 | 华东理工大学 | A kind of efficient Transpositional mutation system and construction method |
| CN112652222A (en) * | 2020-04-30 | 2021-04-13 | 华南农业大学 | Intra-ovarian fertilization process of female silkworm moths and model display method of ovaries |
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| CN1717486A (en) * | 2002-11-29 | 2006-01-04 | 贝林格尔英格海姆法玛两合公司 | Expression vector, methods for the production of heterologous gene products and for the selection of recombinant cells producing high levels of such products |
| CN1912140A (en) * | 2006-08-07 | 2007-02-14 | 浙江大学 | Faultless testing method of transgenosis insect fluorescence gene expression relative volume |
| CN101151378A (en) * | 2005-03-30 | 2008-03-26 | 奥林巴斯株式会社 | Predermined site luminescence measuring method, predetermined site luminenscence measuring apparatus, expression amount measuring method, and measuring apparatus |
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| CN1717486A (en) * | 2002-11-29 | 2006-01-04 | 贝林格尔英格海姆法玛两合公司 | Expression vector, methods for the production of heterologous gene products and for the selection of recombinant cells producing high levels of such products |
| CN101151378A (en) * | 2005-03-30 | 2008-03-26 | 奥林巴斯株式会社 | Predermined site luminescence measuring method, predetermined site luminenscence measuring apparatus, expression amount measuring method, and measuring apparatus |
| CN1912140A (en) * | 2006-08-07 | 2007-02-14 | 浙江大学 | Faultless testing method of transgenosis insect fluorescence gene expression relative volume |
| CN102876706A (en) * | 2012-08-01 | 2013-01-16 | 中国科学院微生物研究所 | Method for high-throughput screening recombinant trichoderma reesei for efficiently expressing foreign protein |
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| CN112652222A (en) * | 2020-04-30 | 2021-04-13 | 华南农业大学 | Intra-ovarian fertilization process of female silkworm moths and model display method of ovaries |
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