Transferência gênica em células espermáticas de Mus musculus e Ramdia quelen
Autor(a) principal: | |
---|---|
Data de Publicação: | 2009 |
Tipo de documento: | Tese |
Idioma: | por |
Título da fonte: | Repositório Institucional da UFPel - Guaiaca |
Texto Completo: | http://guaiaca.ufpel.edu.br/handle/123456789/1270 |
Resumo: | Transgenic animals have been used as biological models in studies of the genes functions and their mechanisms of action, as well as to improve animal production. Researchers are trying to produce transgenic animals that will be organs donors in xenotransplants. Another use of the transgenic animal is in the production of recombinant proteins for pharmaceutical interest, starting from several tissues and corporal fluids of different animal species. Using TMGT (Testis Mediated Gene Transfer), the efficiency of pEGFP transgene transmission in mice using non surgical TMGT was evaluated, without epididymis electroporation; using transfectants as DMSO, liposomes, and for the first time the DMA. To evaluate the efficiency of non surgical TMGT in F0 the EGFP expression was evaluated in vivo and detection in genome was conducted by PCR analysis. Moreover, we evaluated which transfectants were more efficient in transgene transmission and if it induce histological damage in testis, by histological analysis. EGFP expression was not detected in F0 through the ultraviolet light.The result of the PCR analysis shows that liposomes and DMSO were the best transfectants for pEGFP in F0. The histological analysis shows that injections of DMSO with exogenous DNA could affect the development of the germ cell of seminal tubules. The purpose about SMGT was to evaluate the interaction of the spermatozoa of silver catfish with pEGFP vector. It was observed that the semen after three washes in isosmotic solution and at 1000 x g centrifugation could eliminate seminal plasma proteins and preserve cellular motility. The time of action of DNase in the seminal plasma was 30 minutes, the temperature of action of DNase ranged between 33-53°C and its inhibition was detected at 70°C. In the presence of EDTA 30mM the activity of DNase was inhibited. Through PCR it was detected that in the DNA of the silver catfish s spermatozoids, the amplicon of EGFP at different concentrations of pEGFP vector (5-100 ng/106 spermatozoa). We demonstrate that spermatozoa of the silver catfish need to be washed to remove seminal plasma before contact with exogenous DNA, after several washes exogenous DNA was internalized in spermatozoa. |
id |
UFPL_3b4b655096e80c003a4149fed815dbab |
---|---|
oai_identifier_str |
oai:guaiaca.ufpel.edu.br:123456789/1270 |
network_acronym_str |
UFPL |
network_name_str |
Repositório Institucional da UFPel - Guaiaca |
repository_id_str |
|
spelling |
2014-08-20T13:32:54Z2010-05-072014-08-20T13:32:54Z2009-12-28AMARAL, Marta Gonçalves. Genic transfer in sperm cells in Mus musculus and Ramdia quelem. 2009. 77 f. Tese (Doutorado em Biotecnologia) - Universidade Federal de Pelotas, Pelotas, 2009.http://guaiaca.ufpel.edu.br/handle/123456789/1270Transgenic animals have been used as biological models in studies of the genes functions and their mechanisms of action, as well as to improve animal production. Researchers are trying to produce transgenic animals that will be organs donors in xenotransplants. Another use of the transgenic animal is in the production of recombinant proteins for pharmaceutical interest, starting from several tissues and corporal fluids of different animal species. Using TMGT (Testis Mediated Gene Transfer), the efficiency of pEGFP transgene transmission in mice using non surgical TMGT was evaluated, without epididymis electroporation; using transfectants as DMSO, liposomes, and for the first time the DMA. To evaluate the efficiency of non surgical TMGT in F0 the EGFP expression was evaluated in vivo and detection in genome was conducted by PCR analysis. Moreover, we evaluated which transfectants were more efficient in transgene transmission and if it induce histological damage in testis, by histological analysis. EGFP expression was not detected in F0 through the ultraviolet light.The result of the PCR analysis shows that liposomes and DMSO were the best transfectants for pEGFP in F0. The histological analysis shows that injections of DMSO with exogenous DNA could affect the development of the germ cell of seminal tubules. The purpose about SMGT was to evaluate the interaction of the spermatozoa of silver catfish with pEGFP vector. It was observed that the semen after three washes in isosmotic solution and at 1000 x g centrifugation could eliminate seminal plasma proteins and preserve cellular motility. The time of action of DNase in the seminal plasma was 30 minutes, the temperature of action of DNase ranged between 33-53°C and its inhibition was detected at 70°C. In the presence of EDTA 30mM the activity of DNase was inhibited. Through PCR it was detected that in the DNA of the silver catfish s spermatozoids, the amplicon of EGFP at different concentrations of pEGFP vector (5-100 ng/106 spermatozoa). We demonstrate that spermatozoa of the silver catfish need to be washed to remove seminal plasma before contact with exogenous DNA, after several washes exogenous DNA was internalized in spermatozoa.Os animais transgênicos vêm sendo empregados como modelos biológicos em estudos das funções dos genes e dos seus mecanismos de ação, bem como para melhorar a produção animal. Pesquisadores vêm tentando produzir animais transgênicos que serão doadores de órgãos em xenotransplantes. Outra utilização da transgênese animal é a produção de proteínas recombinantes de interesse farmacêutico a partir de diversos tecidos e fluidos corporais de diferentes espécies de animais. Em relação à TMGT (Testis Mediated Gene Transfer) foi avaliada a eficiência da transmissão do transgene EGFP em camundongos, utilizando a TMGT não cirúrgica, sem o uso de eletroporação no epidídimo; utilizando transfectantes como o DMSO, lipossomos, e pela primeira vez o DMA. A detecção da expressão do EGFP foi avaliada in vivo na F0 e por PCR, para comprovar a eficiência da TMGT não cirúrgica. Também foi analisado qual dos transfectantes propiciou a maior taxa de transmissão e se eles causaram danos histológicos aos testículos, através de análise histológica. Não foi detectada a expressão de EGFP, através da luz ultravioeta na F0. Os resultados da análise de PCR demonstraram que o lipossomo e o DMSO foram os melhores transfectantes do pEGFP na F0. A análise histológica demonstrou que a injeção de DMSO com o DNA exógeno, pode comprometer o desenvolvimento das células germinativas do túbulo seminal. O objetivo em relação à SMGT (Sperm-mediated gene transfer) foi avaliar a interação dos espermatozóides de silver catfish (Rhandia quelen) com o vetor pEGFP. Foi observado que o sêmen após três lavagens em solução isosmótica e centrifugadas à 1000 x g, eliminaram as proteínas do plasma seminal e preservaram a motilidade celular. O tempo de atividade da DNase no plasma seminal foi de 30 minutos, a temperatura de atividade da DNase variou entre 33-53°C e sua inativação ocorreu aos 70°C. Na presença de EDTA 30mM a atividade da DNase foi inibida. Através da PCR foi detectada a presença do pEGFP no DNA dos espermatozoides do silver catfish, que incorporaram o vetor em diferentes concentrações (5-100 ng/106 espermatozoides). Concluímos que os espermatozoides do silver catfish precisam ser lavados para retirada do da DNase do plasma seminal antes de entrar em contato com o DNA exógeno, e que após as lavagens ocorreu a internalização do DNA exógeno no espermatozoide.application/pdfporUniversidade Federal de PelotasPrograma de Pós-Graduação em BiotecnologiaUFPelBRBiotecnologiaSpermatozoaExogenous DNANon surgical TMGTDNaseMammalsFishEspermatozóidesDNA exógenoTMGT não cirúrgicaDNaseMamíferosPeixesCNPQ::CIENCIAS BIOLOGICAS::GENETICA::GENETICA ANIMALTransferência gênica em células espermáticas de Mus musculus e Ramdia quelenGenic transfer in sperm cells in Mus musculus and Ramdia queleminfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/doctoralThesishttp://lattes.cnpq.br/5864967684107584http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4783390T8Deschamps, João CarlosAmaral, Marta Gonçalvesinfo:eu-repo/semantics/openAccessreponame:Repositório Institucional da UFPel - Guaiacainstname:Universidade Federal de Pelotas (UFPEL)instacron:UFPELORIGINALtese_marta_amaral.pdfapplication/pdf872749http://guaiaca.ufpel.edu.br/xmlui/bitstream/123456789/1270/1/tese_marta_amaral.pdf441aef851eaec2633ad2f7530bf3c07cMD51open accessTEXTtese_marta_amaral.pdf.txttese_marta_amaral.pdf.txtExtracted Texttext/plain104104http://guaiaca.ufpel.edu.br/xmlui/bitstream/123456789/1270/2/tese_marta_amaral.pdf.txt1cf7a825749ad2ca7b63b3a4ec3d5f44MD52open accessTHUMBNAILtese_marta_amaral.pdf.jpgtese_marta_amaral.pdf.jpgGenerated Thumbnailimage/jpeg1784http://guaiaca.ufpel.edu.br/xmlui/bitstream/123456789/1270/3/tese_marta_amaral.pdf.jpgb93ab0a5394a9526badabe5a8ea56e4dMD53open access123456789/12702019-08-23 10:48:10.72open accessoai:guaiaca.ufpel.edu.br:123456789/1270Repositório InstitucionalPUBhttp://repositorio.ufpel.edu.br/oai/requestrippel@ufpel.edu.br || repositorio@ufpel.edu.br || aline.batista@ufpel.edu.bropendoar:2019-08-23T13:48:10Repositório Institucional da UFPel - Guaiaca - Universidade Federal de Pelotas (UFPEL)false |
dc.title.por.fl_str_mv |
Transferência gênica em células espermáticas de Mus musculus e Ramdia quelen |
dc.title.alternative.eng.fl_str_mv |
Genic transfer in sperm cells in Mus musculus and Ramdia quelem |
title |
Transferência gênica em células espermáticas de Mus musculus e Ramdia quelen |
spellingShingle |
Transferência gênica em células espermáticas de Mus musculus e Ramdia quelen Amaral, Marta Gonçalves Spermatozoa Exogenous DNA Non surgical TMGT DNase Mammals Fish Espermatozóides DNA exógeno TMGT não cirúrgica DNase Mamíferos Peixes CNPQ::CIENCIAS BIOLOGICAS::GENETICA::GENETICA ANIMAL |
title_short |
Transferência gênica em células espermáticas de Mus musculus e Ramdia quelen |
title_full |
Transferência gênica em células espermáticas de Mus musculus e Ramdia quelen |
title_fullStr |
Transferência gênica em células espermáticas de Mus musculus e Ramdia quelen |
title_full_unstemmed |
Transferência gênica em células espermáticas de Mus musculus e Ramdia quelen |
title_sort |
Transferência gênica em células espermáticas de Mus musculus e Ramdia quelen |
author |
Amaral, Marta Gonçalves |
author_facet |
Amaral, Marta Gonçalves |
author_role |
author |
dc.contributor.authorLattes.por.fl_str_mv |
http://lattes.cnpq.br/5864967684107584 |
dc.contributor.advisorLattes.por.fl_str_mv |
http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4783390T8 |
dc.contributor.advisor1.fl_str_mv |
Deschamps, João Carlos |
dc.contributor.author.fl_str_mv |
Amaral, Marta Gonçalves |
contributor_str_mv |
Deschamps, João Carlos |
dc.subject.eng.fl_str_mv |
Spermatozoa Exogenous DNA Non surgical TMGT DNase Mammals Fish |
topic |
Spermatozoa Exogenous DNA Non surgical TMGT DNase Mammals Fish Espermatozóides DNA exógeno TMGT não cirúrgica DNase Mamíferos Peixes CNPQ::CIENCIAS BIOLOGICAS::GENETICA::GENETICA ANIMAL |
dc.subject.por.fl_str_mv |
Espermatozóides DNA exógeno TMGT não cirúrgica DNase Mamíferos Peixes |
dc.subject.cnpq.fl_str_mv |
CNPQ::CIENCIAS BIOLOGICAS::GENETICA::GENETICA ANIMAL |
description |
Transgenic animals have been used as biological models in studies of the genes functions and their mechanisms of action, as well as to improve animal production. Researchers are trying to produce transgenic animals that will be organs donors in xenotransplants. Another use of the transgenic animal is in the production of recombinant proteins for pharmaceutical interest, starting from several tissues and corporal fluids of different animal species. Using TMGT (Testis Mediated Gene Transfer), the efficiency of pEGFP transgene transmission in mice using non surgical TMGT was evaluated, without epididymis electroporation; using transfectants as DMSO, liposomes, and for the first time the DMA. To evaluate the efficiency of non surgical TMGT in F0 the EGFP expression was evaluated in vivo and detection in genome was conducted by PCR analysis. Moreover, we evaluated which transfectants were more efficient in transgene transmission and if it induce histological damage in testis, by histological analysis. EGFP expression was not detected in F0 through the ultraviolet light.The result of the PCR analysis shows that liposomes and DMSO were the best transfectants for pEGFP in F0. The histological analysis shows that injections of DMSO with exogenous DNA could affect the development of the germ cell of seminal tubules. The purpose about SMGT was to evaluate the interaction of the spermatozoa of silver catfish with pEGFP vector. It was observed that the semen after three washes in isosmotic solution and at 1000 x g centrifugation could eliminate seminal plasma proteins and preserve cellular motility. The time of action of DNase in the seminal plasma was 30 minutes, the temperature of action of DNase ranged between 33-53°C and its inhibition was detected at 70°C. In the presence of EDTA 30mM the activity of DNase was inhibited. Through PCR it was detected that in the DNA of the silver catfish s spermatozoids, the amplicon of EGFP at different concentrations of pEGFP vector (5-100 ng/106 spermatozoa). We demonstrate that spermatozoa of the silver catfish need to be washed to remove seminal plasma before contact with exogenous DNA, after several washes exogenous DNA was internalized in spermatozoa. |
publishDate |
2009 |
dc.date.issued.fl_str_mv |
2009-12-28 |
dc.date.available.fl_str_mv |
2010-05-07 2014-08-20T13:32:54Z |
dc.date.accessioned.fl_str_mv |
2014-08-20T13:32:54Z |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/doctoralThesis |
format |
doctoralThesis |
status_str |
publishedVersion |
dc.identifier.citation.fl_str_mv |
AMARAL, Marta Gonçalves. Genic transfer in sperm cells in Mus musculus and Ramdia quelem. 2009. 77 f. Tese (Doutorado em Biotecnologia) - Universidade Federal de Pelotas, Pelotas, 2009. |
dc.identifier.uri.fl_str_mv |
http://guaiaca.ufpel.edu.br/handle/123456789/1270 |
identifier_str_mv |
AMARAL, Marta Gonçalves. Genic transfer in sperm cells in Mus musculus and Ramdia quelem. 2009. 77 f. Tese (Doutorado em Biotecnologia) - Universidade Federal de Pelotas, Pelotas, 2009. |
url |
http://guaiaca.ufpel.edu.br/handle/123456789/1270 |
dc.language.iso.fl_str_mv |
por |
language |
por |
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 Pelotas |
dc.publisher.program.fl_str_mv |
Programa de Pós-Graduação em Biotecnologia |
dc.publisher.initials.fl_str_mv |
UFPel |
dc.publisher.country.fl_str_mv |
BR |
dc.publisher.department.fl_str_mv |
Biotecnologia |
publisher.none.fl_str_mv |
Universidade Federal de Pelotas |
dc.source.none.fl_str_mv |
reponame:Repositório Institucional da UFPel - Guaiaca instname:Universidade Federal de Pelotas (UFPEL) instacron:UFPEL |
instname_str |
Universidade Federal de Pelotas (UFPEL) |
instacron_str |
UFPEL |
institution |
UFPEL |
reponame_str |
Repositório Institucional da UFPel - Guaiaca |
collection |
Repositório Institucional da UFPel - Guaiaca |
bitstream.url.fl_str_mv |
http://guaiaca.ufpel.edu.br/xmlui/bitstream/123456789/1270/1/tese_marta_amaral.pdf http://guaiaca.ufpel.edu.br/xmlui/bitstream/123456789/1270/2/tese_marta_amaral.pdf.txt http://guaiaca.ufpel.edu.br/xmlui/bitstream/123456789/1270/3/tese_marta_amaral.pdf.jpg |
bitstream.checksum.fl_str_mv |
441aef851eaec2633ad2f7530bf3c07c 1cf7a825749ad2ca7b63b3a4ec3d5f44 b93ab0a5394a9526badabe5a8ea56e4d |
bitstream.checksumAlgorithm.fl_str_mv |
MD5 MD5 MD5 |
repository.name.fl_str_mv |
Repositório Institucional da UFPel - Guaiaca - Universidade Federal de Pelotas (UFPEL) |
repository.mail.fl_str_mv |
rippel@ufpel.edu.br || repositorio@ufpel.edu.br || aline.batista@ufpel.edu.br |
_version_ |
1801846968709808128 |