O transposon piggyBac: quantificando sua mobilização
Autor(a) principal: | |
---|---|
Data de Publicação: | 2015 |
Tipo de documento: | Dissertação |
Idioma: | por |
Título da fonte: | Manancial - Repositório Digital da UFSM |
Texto Completo: | http://repositorio.ufsm.br/handle/1/5337 |
Resumo: | In this work we presented the idea to perform excision assays using the piggyBac transposable element as enzyme supplier and the inverted terminal sequences of the element, both necessary for mobilization of a transposable element. Drosophila S2 cells were electroporated to perform insertion of two different plasmids in the cytoplasm of cells, a plasmid carrying the terminal inverted repeats of piggyBac element flanking a GFP gene and other with the transposase coding sequence enzyme which recognizes the terminal inverted repeats, excise of the region where the element is and insert it into another locus. This is a vector-helper system, in which a fragment is excised from a plasmid with the help of the transposase located in the other. Conventional PCR was used to verify excision events showing a 200bp amplification region where the fragment was excised and a region 3kb amplification reagion at times when the fragment was full, ie, it has not mobilized. The qPCR technique was used to quantify the excision of this fragment, carrying out comparisons of the amount of plasmid DNA recovered from the S2 cells after the end of experiment with serial dilutions of the original plasmids carrying the ITRs, which was used as standard. The results showed that the technique involving electroporation and qPCR is feasible and can be used to quantify mobilization of transposable elements. Paralleling with existing tools for this type of quantification, qPCR shows up as a very sensitive technique of detection mobilization, as well as a low cost technique budget. |
id |
UFSM-20_8f32ac28ee96dff3c83f82250db1558f |
---|---|
oai_identifier_str |
oai:repositorio.ufsm.br:1/5337 |
network_acronym_str |
UFSM-20 |
network_name_str |
Manancial - Repositório Digital da UFSM |
repository_id_str |
3913 |
spelling |
2016-09-212016-09-212015-05-05KAMINSKI, Valéria de Lima. A new way to quantify transposon mobilization using piggyBac as model. 2015. 38 f. Dissertação (Mestrado em Ciencias Biológicas) - Universidade Federal de Santa Maria, Santa Maria, 2015.http://repositorio.ufsm.br/handle/1/5337In this work we presented the idea to perform excision assays using the piggyBac transposable element as enzyme supplier and the inverted terminal sequences of the element, both necessary for mobilization of a transposable element. Drosophila S2 cells were electroporated to perform insertion of two different plasmids in the cytoplasm of cells, a plasmid carrying the terminal inverted repeats of piggyBac element flanking a GFP gene and other with the transposase coding sequence enzyme which recognizes the terminal inverted repeats, excise of the region where the element is and insert it into another locus. This is a vector-helper system, in which a fragment is excised from a plasmid with the help of the transposase located in the other. Conventional PCR was used to verify excision events showing a 200bp amplification region where the fragment was excised and a region 3kb amplification reagion at times when the fragment was full, ie, it has not mobilized. The qPCR technique was used to quantify the excision of this fragment, carrying out comparisons of the amount of plasmid DNA recovered from the S2 cells after the end of experiment with serial dilutions of the original plasmids carrying the ITRs, which was used as standard. The results showed that the technique involving electroporation and qPCR is feasible and can be used to quantify mobilization of transposable elements. Paralleling with existing tools for this type of quantification, qPCR shows up as a very sensitive technique of detection mobilization, as well as a low cost technique budget.Neste trabalho apresentamos a ideia de realizar ensaios de excisão utilizando o elemento transponível piggyBac como fornecedor da enzima e das sequências terminais invertidas do elemento, ambos necessários para mobilização. Células S2 de Drosophila melanogaster foram eletroporadas para que houvesse inserção de dois diferentes plasmídeos no citoplasma das células, um plasmídeo portando as repetições terminais invertidas do elemento piggyBac flanqueando um gene GFP e o outro com a sequência codificadora da enzima transposase, a qual reconhece as repetições terminais invertidas e excisa o elemento da região onde está inserido, num sistema vector-helper, em que um fragmento é excisado de um plasmídeo com ajuda da transposase localizada no outro. PCR convencional foi usado para verificar os eventos de excisão, mostrando uma região de amplificação de 200pb nos casos de excisão do fragmento e uma região amplicada de 3kb, nas ocasiões em que o fragmento ficou inteiro, ou seja, não foi mobilizado. A qPCR foi utilizada para quantificar a excisão desse fragmento, realizando comparações da quantidade de DNA plasmidial recuperado das células S2 após o término do experimento com diluições em série do plasmídeo com as ITRs, que foi utilizado como standard. Os resultados mostraram que a técnica envolvendo eletroporação e qPCR é exequível e pode ser utilizada para quantificar mobilização de elementos transponíveis. Fazendo um paralelo com as ferramentas já existentes para esse tipo de quantificação, qPCR mostra-se como uma técnica bastante sensível de detecção de mobilização, bem como uma técnica de baixo custo orçamentário.Conselho Nacional de Desenvolvimento Científico e Tecnológicoapplication/pdfporUniversidade Federal de Santa MariaPrograma de Pós-Graduação em Biodiversidade AnimalUFSMBRCiências BiológicasCélulas S2PiggyBacExcisãoEletroporaçãoQPCRS2 cell lineagePiggyBacExcisionElectroporationQPCRCNPQ::CIENCIAS BIOLOGICASO transposon piggyBac: quantificando sua mobilizaçãoA new way to quantify transposon mobilization using piggyBac as modelinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/masterThesisLoreto, Elgion Lucio da Silvahttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4785575A7Schuch, André Passagliahttp://lattes.cnpq.br/4932611269622766Graichen, Daniel ângelo Sganzerlahttp://lattes.cnpq.br/0162800772752430Gaiesky, Vera Lúcia da Silva Valentehttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4788980U6http://lattes.cnpq.br/8648323421431193Kaminski, Valéria de Lima2000000000064005003003003003001ba859a2-58b7-4e36-9d19-b101c4d2f66973456559-857c-400f-bbf5-abab95aa243f6ee75042-a06b-48e6-a37c-ef9600da01f834585484-9b8f-4f3a-aa42-1998186b71560e0a181f-c3b2-4121-8684-5b4c7669f3bainfo:eu-repo/semantics/openAccessreponame:Manancial - Repositório Digital da UFSMinstname:Universidade Federal de Santa Maria (UFSM)instacron:UFSMORIGINALKAMINSKI, VALERIA DE LIMA.pdfapplication/pdf619434http://repositorio.ufsm.br/bitstream/1/5337/1/KAMINSKI%2c%20VALERIA%20DE%20LIMA.pdfc96e2d9612da1f8ec8df0e2222304204MD51TEXTKAMINSKI, VALERIA DE LIMA.pdf.txtKAMINSKI, VALERIA DE LIMA.pdf.txtExtracted texttext/plain62827http://repositorio.ufsm.br/bitstream/1/5337/2/KAMINSKI%2c%20VALERIA%20DE%20LIMA.pdf.txte37c9f74ba7905a4edb0f1108a7c2744MD52THUMBNAILKAMINSKI, VALERIA DE LIMA.pdf.jpgKAMINSKI, VALERIA DE LIMA.pdf.jpgIM Thumbnailimage/jpeg5058http://repositorio.ufsm.br/bitstream/1/5337/3/KAMINSKI%2c%20VALERIA%20DE%20LIMA.pdf.jpg38ff0fd40cb0795f1df85b2acebe3cb4MD531/53372022-03-17 10:55:04.742oai:repositorio.ufsm.br:1/5337Repositório Institucionalhttp://repositorio.ufsm.br/PUBhttp://repositorio.ufsm.br/oai/requestopendoar:39132022-03-17T13:55:04Manancial - Repositório Digital da UFSM - Universidade Federal de Santa Maria (UFSM)false |
dc.title.por.fl_str_mv |
O transposon piggyBac: quantificando sua mobilização |
dc.title.alternative.eng.fl_str_mv |
A new way to quantify transposon mobilization using piggyBac as model |
title |
O transposon piggyBac: quantificando sua mobilização |
spellingShingle |
O transposon piggyBac: quantificando sua mobilização Kaminski, Valéria de Lima Células S2 PiggyBac Excisão Eletroporação QPCR S2 cell lineage PiggyBac Excision Electroporation QPCR CNPQ::CIENCIAS BIOLOGICAS |
title_short |
O transposon piggyBac: quantificando sua mobilização |
title_full |
O transposon piggyBac: quantificando sua mobilização |
title_fullStr |
O transposon piggyBac: quantificando sua mobilização |
title_full_unstemmed |
O transposon piggyBac: quantificando sua mobilização |
title_sort |
O transposon piggyBac: quantificando sua mobilização |
author |
Kaminski, Valéria de Lima |
author_facet |
Kaminski, Valéria de Lima |
author_role |
author |
dc.contributor.advisor1.fl_str_mv |
Loreto, Elgion Lucio da Silva |
dc.contributor.advisor1Lattes.fl_str_mv |
http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4785575A7 |
dc.contributor.referee1.fl_str_mv |
Schuch, André Passaglia |
dc.contributor.referee1Lattes.fl_str_mv |
http://lattes.cnpq.br/4932611269622766 |
dc.contributor.referee2.fl_str_mv |
Graichen, Daniel ângelo Sganzerla |
dc.contributor.referee2Lattes.fl_str_mv |
http://lattes.cnpq.br/0162800772752430 |
dc.contributor.referee3.fl_str_mv |
Gaiesky, Vera Lúcia da Silva Valente |
dc.contributor.referee3Lattes.fl_str_mv |
http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4788980U6 |
dc.contributor.authorLattes.fl_str_mv |
http://lattes.cnpq.br/8648323421431193 |
dc.contributor.author.fl_str_mv |
Kaminski, Valéria de Lima |
contributor_str_mv |
Loreto, Elgion Lucio da Silva Schuch, André Passaglia Graichen, Daniel ângelo Sganzerla Gaiesky, Vera Lúcia da Silva Valente |
dc.subject.por.fl_str_mv |
Células S2 PiggyBac Excisão Eletroporação QPCR |
topic |
Células S2 PiggyBac Excisão Eletroporação QPCR S2 cell lineage PiggyBac Excision Electroporation QPCR CNPQ::CIENCIAS BIOLOGICAS |
dc.subject.eng.fl_str_mv |
S2 cell lineage PiggyBac Excision Electroporation QPCR |
dc.subject.cnpq.fl_str_mv |
CNPQ::CIENCIAS BIOLOGICAS |
description |
In this work we presented the idea to perform excision assays using the piggyBac transposable element as enzyme supplier and the inverted terminal sequences of the element, both necessary for mobilization of a transposable element. Drosophila S2 cells were electroporated to perform insertion of two different plasmids in the cytoplasm of cells, a plasmid carrying the terminal inverted repeats of piggyBac element flanking a GFP gene and other with the transposase coding sequence enzyme which recognizes the terminal inverted repeats, excise of the region where the element is and insert it into another locus. This is a vector-helper system, in which a fragment is excised from a plasmid with the help of the transposase located in the other. Conventional PCR was used to verify excision events showing a 200bp amplification region where the fragment was excised and a region 3kb amplification reagion at times when the fragment was full, ie, it has not mobilized. The qPCR technique was used to quantify the excision of this fragment, carrying out comparisons of the amount of plasmid DNA recovered from the S2 cells after the end of experiment with serial dilutions of the original plasmids carrying the ITRs, which was used as standard. The results showed that the technique involving electroporation and qPCR is feasible and can be used to quantify mobilization of transposable elements. Paralleling with existing tools for this type of quantification, qPCR shows up as a very sensitive technique of detection mobilization, as well as a low cost technique budget. |
publishDate |
2015 |
dc.date.issued.fl_str_mv |
2015-05-05 |
dc.date.accessioned.fl_str_mv |
2016-09-21 |
dc.date.available.fl_str_mv |
2016-09-21 |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/masterThesis |
format |
masterThesis |
status_str |
publishedVersion |
dc.identifier.citation.fl_str_mv |
KAMINSKI, Valéria de Lima. A new way to quantify transposon mobilization using piggyBac as model. 2015. 38 f. Dissertação (Mestrado em Ciencias Biológicas) - Universidade Federal de Santa Maria, Santa Maria, 2015. |
dc.identifier.uri.fl_str_mv |
http://repositorio.ufsm.br/handle/1/5337 |
identifier_str_mv |
KAMINSKI, Valéria de Lima. A new way to quantify transposon mobilization using piggyBac as model. 2015. 38 f. Dissertação (Mestrado em Ciencias Biológicas) - Universidade Federal de Santa Maria, Santa Maria, 2015. |
url |
http://repositorio.ufsm.br/handle/1/5337 |
dc.language.iso.fl_str_mv |
por |
language |
por |
dc.relation.cnpq.fl_str_mv |
200000000006 |
dc.relation.confidence.fl_str_mv |
400 500 300 300 300 300 |
dc.relation.authority.fl_str_mv |
1ba859a2-58b7-4e36-9d19-b101c4d2f669 73456559-857c-400f-bbf5-abab95aa243f 6ee75042-a06b-48e6-a37c-ef9600da01f8 34585484-9b8f-4f3a-aa42-1998186b7156 0e0a181f-c3b2-4121-8684-5b4c7669f3ba |
dc.rights.driver.fl_str_mv |
info:eu-repo/semantics/openAccess |
eu_rights_str_mv |
openAccess |
dc.format.none.fl_str_mv |
application/pdf |
dc.publisher.none.fl_str_mv |
Universidade Federal de Santa Maria |
dc.publisher.program.fl_str_mv |
Programa de Pós-Graduação em Biodiversidade Animal |
dc.publisher.initials.fl_str_mv |
UFSM |
dc.publisher.country.fl_str_mv |
BR |
dc.publisher.department.fl_str_mv |
Ciências Biológicas |
publisher.none.fl_str_mv |
Universidade Federal de Santa Maria |
dc.source.none.fl_str_mv |
reponame:Manancial - Repositório Digital da UFSM instname:Universidade Federal de Santa Maria (UFSM) instacron:UFSM |
instname_str |
Universidade Federal de Santa Maria (UFSM) |
instacron_str |
UFSM |
institution |
UFSM |
reponame_str |
Manancial - Repositório Digital da UFSM |
collection |
Manancial - Repositório Digital da UFSM |
bitstream.url.fl_str_mv |
http://repositorio.ufsm.br/bitstream/1/5337/1/KAMINSKI%2c%20VALERIA%20DE%20LIMA.pdf http://repositorio.ufsm.br/bitstream/1/5337/2/KAMINSKI%2c%20VALERIA%20DE%20LIMA.pdf.txt http://repositorio.ufsm.br/bitstream/1/5337/3/KAMINSKI%2c%20VALERIA%20DE%20LIMA.pdf.jpg |
bitstream.checksum.fl_str_mv |
c96e2d9612da1f8ec8df0e2222304204 e37c9f74ba7905a4edb0f1108a7c2744 38ff0fd40cb0795f1df85b2acebe3cb4 |
bitstream.checksumAlgorithm.fl_str_mv |
MD5 MD5 MD5 |
repository.name.fl_str_mv |
Manancial - Repositório Digital da UFSM - Universidade Federal de Santa Maria (UFSM) |
repository.mail.fl_str_mv |
|
_version_ |
1801223680819200000 |