{"id":105322,"date":"2018-03-11T10:28:48","date_gmt":"2018-03-11T10:28:48","guid":{"rendered":"https:\/\/www.deberes.net\/tesis\/sin-categoria\/transposones-sb-sleeping-beauty-y-fp-frog-prince-su-aplicabilidad-de-la-transferencia-de-adn-a-celulas-de-peces\/"},"modified":"2018-03-11T10:28:48","modified_gmt":"2018-03-11T10:28:48","slug":"transposones-sb-sleeping-beauty-y-fp-frog-prince-su-aplicabilidad-de-la-transferencia-de-adn-a-celulas-de-peces","status":"publish","type":"post","link":"https:\/\/www.deberes.net\/tesis\/genetica-animal\/transposones-sb-sleeping-beauty-y-fp-frog-prince-su-aplicabilidad-de-la-transferencia-de-adn-a-celulas-de-peces\/","title":{"rendered":"Transposones sb (sleeping beauty) y fp (frog prince): su aplicabilidad de la transferencia de adn a celulas de peces"},"content":{"rendered":"<h2>Tesis doctoral de <strong> Jos\u00e9 Braulio Gallardo Galvez <\/strong><\/h2>\n<p>T\u00edtulo de la tesis: transposones sb (sleeping beauty) y fp (frog prince): su apucabiudad en la transferencia de adn a c\u00e9lulas de peces resumen: la transferencia de adn a c\u00e9lulas o embriones de peces tiene numerosas aplicaciones, tales como el an\u00e1lisis funcional de genes de inter\u00e9s, la obtenci\u00f3n de biorreaclores o la pasible creaci\u00f3n de peces transg\u00e9nicos. En base a los \u00e9xitos obtenidos en ensayos previos al utilizar transposones 7c7 en la transformaci\u00f3n de especies heter\u00f3logas, sobre todo de mam\u00edferos, se estudia el posible papel de transposones tipo 7cf en la mejora de la integraci\u00f3n y expresi\u00f3n de transgenes en el genomio de especies pisc\u00edcolas. Para ello se eval\u00faa la eficiencia de transposici\u00f3n de los sistemas sb y fp en lineas celulares de peces procedentes de especies filogen\u00e9ticamente alejadas (chse-214, rtg-2, bf-2, epc y saf-1), usando para ello los ensayos de escisi\u00f3n y formaci\u00f3n de colonias. Adem\u00e1s se eval\u00faan por ensayos directos la actividad del sistema sb, desarrollado a partir del genoma de salm\u00f3nidos, en la l\u00ednea chse-214 de salm\u00f3n y se determina in vitro la capacidad del sistema sb para desarrollar un sistema de expresi\u00f3n en la l\u00ednea chse-214 de salm\u00f3n de la prote\u00edna mx de lenguado (sofea senegalensis). B\u00e1sicamente se comprueba que las transposasas sb y fp son activas en las cinco l\u00edneas celulares de peces analizadas, en las que presentan un nivel de actividad discreto.No obstante, fp parece m\u00e1s \u00fatil que sb en la transferencia de adn a especies pisc\u00edcolas. En las l\u00edneas de salm\u00f3nidos las transposa sas end\u00f3genas tipo tc1 interfieren con la actividad de sb mediante un mecanismo de complement aci\u00f3n dominante negativa y estas transposa sas end\u00f3genas presentan cierta actividad transposicional en estas l\u00edneas celulares. Se comprueba que la l\u00ednea celular chse-214 se revela como un sistema experimental atractivo para estudiar la interacci\u00f3n entre transposones de la familia 7c f y transposa sas end\u00f3genas. El transpos\u00f3n sb se revela como herramienta \u00fatil para el desarrollo de sistemas de expresi\u00f3n estable de prote\u00ednas recombinantes en las c\u00e9lulas chse-214 los avances en la investigaci\u00f3n sobre transposones est\u00e1n revolucionando la percepci\u00f3n de su naturaleza, de su funci\u00f3n en la regulaci\u00f3n de la expresi\u00f3n g\u00e9nica y de su significado como una fuerza creadora de novedades gen\u00e9ticas. Incluso se ha valorado su potencial evolutivo como \u00abpar\u00e1sitos \u00fatiles\u00bb en la dimensi\u00f3n de linajes, de forma que algunos autores se\u00f1alan la posibilidad de que episodios de alta actividad transposicional, podr\u00edan explicar la teor\u00eda de los equilibrios interrumpidos que sugieren datos paleontol\u00f3gicos. El conocimiento sobre la din\u00e1mica y evoluci\u00f3n de los transposones de peces permitir\u00e1 utilizarlos para desarrollar vectores para la manipulaci\u00f3n gen\u00e9tica, o dise\u00f1ar nuevas aplicaciones biotecnol\u00f3gtcas, incluyendo algunas para acuicultura. fish cells stably expressing exogenous genes nave potential applications in the product!On of fish recombinant proteins, gene-function studies, gene-trapping and the product ion of transgenic fish. However, expr\u00e9s si on of a gene of interest after random integration may be difficutt to predict or control. In the past decade major contri bufa\u00bb ons nave been made in vertebrate-gene trans\u00eder, by using tools derived from dna transposons. Amono, them, the steeping beauty (sb) and frog prince (fp) transposons, derived respectively from fish and frog gen ornes, med\u00edate transposit\u00edon in a large vari\u00e9ty of cells, although with difieren! Efhciency. This study was aimed at assessing the activities of the sb and the fp transposases in fish cefl lines from genefacally d\u00edstant species (chse-214, rtg-2, bf-2, epc and saf-1). Their transposrrjonal ability was evaluated by the plasmid-based excisi\u00f3n assay, the cotony formation assay and the footprint pattems. The results reveal that wh\u00edle both transposases are active \u00abm all cell lines, the faanspos\u00edfaon rates and the precisi\u00f3n of the transposit\u00edon are overall higher with fp than sb. When sb activity was assessed by indirect a p proa ches in various fish cell lines, the salmonid chse-214 cells showed the poorest performance, wh\u00edch was interpreted as due to high lev\u00e9is of tcl-\u00e1ke endogenous transposases that were able to impair sb functionality. To further interpret this peculiar behaviour, in this study we aimed at characterizing sb-mediated integras ons in chse-214 cells, by colony formation assays, along with determining the number and precisi\u00f3n of trans\u00f3ositions that occurred in individual cells, by southern blotrjng and sequencing of the integration sites. The results indicated a slight activity of sb in improving integration rates, although all sequences from single integrations dtd not show expected pattems, which mtght have resutted from either \u00c2\u00a1ilegit\u00edmate recombination or imprecise transposit\u00edon. These findings support the pos si ble interference of sb activity by tne previously detected tc1-like endogenous tranposases that rruglrt be acting as a barr\u00ed er for precise sb-mediated transposit\u00edon. Because sb was obtained from a salmonid genome its use as a vector in heterologous organ\u00edsms may be far more efficient and less likely to cause genontic instab\u00edlity. Finally, in this study aimed at evaluat\u00edng the activity of 58 as an improving gene transfer method, for the estamishment of expression stable cell clones in the salmonid cell line chse-214. The transgene that was chosen was that of the antiviral mx prote\u00edn from the aquacu\u00edtured species senegalese sol\u00e9 (solea senegalensis). The results showed that sb proved effective for the establishment of clones homogeneously and stably expressing the ssmx recombinant protein, whose antiviral activity against sol\u00e9 aquabirnavrrus was successfully tested.<\/p>\n<p>&nbsp;<\/p>\n<h3>Datos acad\u00e9micos de la tesis doctoral \u00ab<strong>Transposones sb (sleeping beauty) y fp (frog prince): su aplicabilidad de la transferencia de adn a celulas de peces<\/strong>\u00ab<\/h3>\n<ul>\n<li><strong>T\u00edtulo de la tesis:<\/strong>\u00a0 Transposones sb (sleeping beauty) y fp (frog prince): su aplicabilidad de la transferencia de adn a celulas de peces <\/li>\n<li><strong>Autor:<\/strong>\u00a0 Jos\u00e9 Braulio Gallardo Galvez <\/li>\n<li><strong>Universidad:<\/strong>\u00a0 M\u00e1laga<\/li>\n<li><strong>Fecha de lectura de la tesis:<\/strong>\u00a0 30\/11\/2010<\/li>\n<\/ul>\n<p>&nbsp;<\/p>\n<h3>Direcci\u00f3n y tribunal<\/h3>\n<ul>\n<li><strong>Director de la tesis<\/strong>\n<ul>\n<li>Mar\u00eda  Del Carmen Alvarez Herrero<\/li>\n<\/ul>\n<\/li>\n<li><strong>Tribunal<\/strong>\n<ul>\n<li>Presidente del tribunal: lLuis Montoliu jose <\/li>\n<li>perry Hackett (vocal)<\/li>\n<li>gonzalo Mart\u00ednez rodr\u00edguez (vocal)<\/li>\n<li>Ana Mar\u00eda G\u00f3mez peris (vocal)<\/li>\n<\/ul>\n<\/li>\n<\/ul>\n<p>&nbsp;<\/p>\n","protected":false},"excerpt":{"rendered":"<p>Tesis doctoral de Jos\u00e9 Braulio Gallardo Galvez T\u00edtulo de la tesis: transposones sb (sleeping beauty) y fp (frog prince): su 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