ES2300200A1 - Transposon hsmar2 and use thereof in the generation of vectors that can be used in somatic gene therapy - Google Patents
Transposon hsmar2 and use thereof in the generation of vectors that can be used in somatic gene therapy Download PDFInfo
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- ES2300200A1 ES2300200A1 ES200602688A ES200602688A ES2300200A1 ES 2300200 A1 ES2300200 A1 ES 2300200A1 ES 200602688 A ES200602688 A ES 200602688A ES 200602688 A ES200602688 A ES 200602688A ES 2300200 A1 ES2300200 A1 ES 2300200A1
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
- A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
- A61K48/0066—Manipulation of the nucleic acid to modify its expression pattern, e.g. enhance its duration of expression, achieved by the presence of particular introns in the delivered nucleic acid
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
- C12N15/90—Stable introduction of foreign DNA into chromosome
- C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
- C12N15/907—Stable introduction of foreign DNA into chromosome using homologous recombination in mammalian cells
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
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- C12N9/22—Ribonucleases RNAses, DNAses
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- C12N2800/00—Nucleic acids vectors
- C12N2800/90—Vectors containing a transposable element
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Abstract
Description
Transposón Hsmar2 y su uso en la generación de vectores útiles en terapia génica somática.Transposón Hsmar2 and its use in the generation of useful vectors in somatic gene therapy.
La invención se refiere a la generación del primer transposón activo humano de ADN, el transposón Hsmar2, así como al análisis de su actividad en células de mamífero y en particular en células humanas. Concretamente, la invención se refiere a la caracterización funcional de Hsmar2 y a su capacidad de transponer fragmentos de ADN que se encuentran flanqueados por las secuencias TR de Hsmar2 de un genoma donante a un genoma receptor.The invention relates to the generation of the first human active DNA transposon, the Hsmar2 transposon, as well as to the analysis of its activity in mammalian cells and in particular in human cells. Specifically, the invention relates to the functional characterization of Hsmar2 and its ability to transpose DNA fragments that are flanked by the Hsmar2 TR sequences from a donor genome to a recipient genome.
Las transposasas son enzimas mobilizadores de ADN, destacando los de la superfamilia Tc1/mariner por su amplia distribución en animales, incluyendo insectos, vertebrados y mamíferos. La estructura de los transposones mariner es simple pues están formados por una única pauta de lectura abierta de unos 1300 pares de bases flanqueada por dos secuencias terminales (ITR = Inverted Terminal Repeats). Hasta el momento, sólo se han reportado dos transposones activos: Mos1 (de Drosophila mauritiana) y Himar1 (de Haemotobia irritans). Así, aunque se han descrito otros transposones en distintas especies, todos contienen mutaciones inactivantes múltiples debido a fenómenos de inactivación vertical (1).Transposases are DNA mobilizing enzymes, highlighting those of the Tc1 / mariner superfamily for their wide distribution in animals, including insects, vertebrates and mammals. The structure of the mariner transposons is simple because they are formed by a single open reading pattern of about 1300 base pairs flanked by two terminal sequences (ITR = Inverted Terminal Repeats). So far, only two active transposons have been reported: Mos1 (from Drosophila mauritiana ) and Himar1 (from Haemotobia irritans ). Thus, although other transposons have been described in different species, all contain multiple inactivating mutations due to vertical inactivation phenomena (1).
Los transposones mariner utilizan un mecanismo de "cortar y pegar" en el que se excinde el transposón de su localización original y se inserta en nuevas localizaciones del genoma. Para llevar a cabo el proceso sólo se necesitan dos elementos: una transposasa activa in trans y un fragmento de ADN flanqueado por dos secuencias ITR. Los fragmentos mobilizados se integran siempre en un dinuclueótido TA, el cual se duplica después de la inserción. Mediante estudios de transposición in vitro con transposasas purificadas se sabe que el proceso de transposición es independiente de factores nucleares (2). Sin embargo, la eficiencia final de transposición puede ser aumentada o disminuida por factores celulares específicos y así las transposasas descritas anteriormente muestran un baja eficiencia en mamíferos.Mariner transposons use a "cut and paste" mechanism in which the transposon is lost from its original location and inserted into new genome locations. To carry out the process, only two elements are needed: an active transposase in trans and a DNA fragment flanked by two ITR sequences. The mobilized fragments are always integrated into a TA dinuclueotide, which doubles after insertion. Through in vitro transposition studies with purified transposases it is known that the transposition process is independent of nuclear factors (2). However, the final efficiency of transposition can be increased or decreased by specific cellular factors and thus the transposases described above show low efficiency in mammals.
Hasta el momento se han descrito dos secuencias consensus de transposones mariner humanos. Ambas secuencias Hsmar1 (3) y Hsmar2 (4) se detectaron por análisis computacional del genoma humano. La secuencia consensus inicial de Hsmar2 fue posteriormente refinada, y consta de dos secuencias ITR de 32 pb, y una secuencia de 1301 pares de bases (pb) que codifica para una transposasa de 351 aminoácidos que muestra un 38% de homología con otros transposones mariners de la misma familia (5) y que contiene en las posiciones (Asp_{160}Asp_{254}Asp_{289}), el dominio acídico AspAsp(_{34/35})Asp característico de los transposones mariner.So far, two consensus sequences of human mariner transposons have been described. Both sequences Hsmar1 (3) and Hsmar2 (4) were detected by computational analysis of the human genome. The initial consensus sequence of Hsmar2 was subsequently refined, and consists of two 32 bp ITR sequences, and a 1301 base pair (bp) sequence that encodes a 351 amino acid transposase that shows 38% homology with other mariner transposons of the same family (5) and containing in the positions (Asp_ {160} Asp_ {254} Asp_ {289}), the acidic domain AspAsp (34/35}) Asp characteristic of mariner transposons.
Utilizando la secuencia consensus de Hsmar2 (número de acceso=U49974). se han detectado por PRINS (prime in situ labelling) más de 100 secuencias Hsmar2-like en el genoma humano (6). Sin embargo, todas las copias analizadas contienen numerosas mutaciones, deleciones, pequeñas y grandes inserciones incluso de elementos Alu. Las copias tienen una homología de 85-90% con la secuencia consensus y una media de 12 mutaciones por kilobase. Estos datos indican que las copias Hsmar2-like son el remanente inactivo de un elemento mariner que entró en el linaje humano hace unos 80 millones de años.Using the consensus sequence of Hsmar2 (access number = U49974). more than 100 Hsmar2- like sequences have been detected by PRINS (prime in situ labelling) in the human genome (6). However, all the analyzed copies contain numerous mutations, deletions, small and large insertions even of Alu elements. The copies have a homology of 85-90% with the consensus sequence and an average of 12 mutations per kilobase. These data indicate that Hsmar2- like copies are the inactive remnant of a mariner element that entered the human lineage about 80 million years ago.
Asimismo, se ha publicado recientemente la reconstrucción de una transposasa mariner activa "Sleeping Beauty" a partir de una secuencia consensus de los Salmónidos Salmo salar y Oncorhynchus mykiss, mediante la eliminación por mutagénesis dirigida de las mutaciones inactivantes de su secuencia (7). Esta Sleeping Beauty de novo muestra una elevada eficiencia en determinados tipos celulares humanos. Aún y así es importante resaltar que aunque los transposones mariner son relativamente autónomos, su actividad y eficiencia está influenciada por la presencia/ausencia de factores celulares específicos. Por ello es posible que el sistema más eficiente para mediar un proceso integrativo en mamíferos, y especialmente en células humanas, sea mediante la utilización de una transposasa humana como la Hsmar2. Sin embargo, debido a que todos los transposones Hsmar2 presentes en el genoma humano contienen numerosas mutaciones inactivantes es necesario generar primero in vitro un transposón que contenga exactamente la secuencia nucleotídica de la secuencia consensus (4) y segundo analizar en células humanas que el transposón es activo y capaz de transponer fragmentos de ADN flanqueados por las secuencias ITR de Hsmar2 entre un genoma donante y un genoma receptor.Likewise, the reconstruction of an active mariner transposase "Sleeping Beauty" from a consensus sequence of Salmo salar Salmonids and Oncorhynchus mykiss has recently been published, by elimination by directed mutagenesis of inactivating mutations of its sequence (7). This Sleeping Beauty de novo shows high efficiency in certain human cell types. Even so, it is important to highlight that although mariner transposons are relatively autonomous, their activity and efficiency is influenced by the presence / absence of specific cellular factors. Therefore, it is possible that the most efficient system to mediate an integrative process in mammals, and especially in human cells, is through the use of a human transposase such as Hsmar2 . However, because all the Hsmar2 transposons present in the human genome contain numerous inactivating mutations, it is necessary to first generate in vitro a transposon that contains exactly the nucleotide sequence of the consensus sequence (4) and second to analyze in human cells that the transposon is active and capable of transposing DNA fragments flanked by the ITR sequences of Hsmar2 between a donor genome and a recipient genome.
La capacidad de integrar secuencias de ADN en el genoma celular tiene un gran interés en el campo de la terapia génica y mas específicamente para el tratamiento de enfermedades crónicas para las cuales es necesaria la expresión permanente del gen terapéutico. Actualmente la expresión permanente se consigue mediante la utilización de vectores integrativos como los virus adenoasociados y los retrovirus ya sean derivados de MMLV (virus de la leucemia murina Moloney) o de HIV (virus del sida). Sin embargo, numerosos grupos de investigación consideran muy conveniente utilizar adenovirus como vectores gracias a que infectan eficientemente una amplia variedad de tipos celulares (quiescentes y en división) y a tener una capacidad máxima de 36 Kb lo cual resulta ventajoso respecto a otros vectores porque la capacidad máxima de los otros vectores virales existentes, que tienen una elevada eficiencia, varía entre las 3'5 kilobases y las 12 kilobases. Por tanto, este vector viral multiplica por 3-10 el tamaño de los genes terapéuticos que puede contener en su genoma. Sin embargo, los adenovirus no se integran y para obtener una expresión prolongada del transgén es necesario administrar el vector en repetidas dosis, lo que genera una respuesta inmune contra el vector y por lo tanto, conlleva la no expresión del transgén en administraciones posteriores (8). Una solución se basa en generar adenovirus con capacidad integrativa que eviten la necesidad de readministrar el vector. La utilización de transposones mariner para generar adenovirus integrativos no es nueva. Ya en 1998 se utilizó una de las dos transposasas activas, Himar1, y se demostró que mediaba procesos de integración en células de mamíferos cuando se utilizaba en un adenovirus (9). Posteriormente, se ha utilizado Sleeping Beauty (SB) (otro transposón mariner) para generar adenovirus integrativos (10). Sin embargo, ninguno de estos transposones es de mamífero, y presentan una baja eficiencia en la integración de secuencias dentro del genoma humano in vivo (alrededor del 3-5%), incluso en tipos celulares de alta tasa de división celular como hepatocitos (11) y keratinocitos (12). Es por ello, que la utilización del transposón Hsmar2 podría aumentar el potencial terapéutico en humanos de adenovirus integrativos y ser utilizado en estrategias de terapia génica para el tratamiento de enfermedades crónicas.The ability to integrate DNA sequences into the cell genome is of great interest in the field of gene therapy and more specifically for the treatment of chronic diseases for which permanent expression of the therapeutic gene is necessary. Currently, permanent expression is achieved through the use of integrative vectors such as adeno-associated viruses and retroviruses, whether derived from MMLV (Moloney murine leukemia virus) or HIV (AIDS virus). However, many research groups consider it very convenient to use adenoviruses as vectors because they efficiently infect a wide variety of cell types (quiescent and in division) and have a maximum capacity of 36 Kb which is advantageous compared to other vectors because the capacity Maximum of the other existing viral vectors, which have a high efficiency, varies between 3.5 kilobases and 12 kilobases. Therefore, this viral vector multiplies by 3-10 the size of the therapeutic genes that it can contain in its genome. However, adenoviruses do not integrate and to obtain prolonged expression of the transgene it is necessary to administer the vector in repeated doses, which generates an immune response against the vector and therefore, leads to non-expression of the transgene in subsequent administrations (8 ). One solution is based on generating adenovirus with integrative capacity that avoid the need to re-administer the vector. The use of mariner transposons to generate integrative adenoviruses is not new. Already in 1998, one of the two active transposases, Himar1 , was used and it was shown that it mediated integration processes in mammalian cells when used in an adenovirus (9). Subsequently, Sleeping Beauty (SB) (another mariner transposon) has been used to generate integrative adenoviruses (10). However, none of these transposons is mammalian, and have a low efficiency in the integration of sequences within the human genome in vivo (about 3-5%), even in cell types of high cell division rate such as hepatocytes (11 ) and keratinocytes (12). That is why the use of the Hsmar2 transposon could increase the therapeutic potential in humans of integrative adenovirus and be used in gene therapy strategies for the treatment of chronic diseases.
En la actualidad se conoce un número muy reducido de transposones de ADN activos, la mayoría de los cuales son de invertebrados. Tan sólo se conoce un transposón de ADN de la familia mariner que sea activo en vertebrados (Sleeping Beauty, de Salmónidos), pero hay ningún transposón de mamíferos, incluidos los humanos. Es importante resaltar que aunque los transposones mariner son relativamente autónomos, su actividad y eficiencia está influenciada por la presencia/ausencia de factores celulares específicos. Por ello pensamos que el transposón más eficiente para mediar un proceso integrativo en mamíferos, y especialmente en células humanas, sería mediante la utilización de una transposasa humana, y más específicamente Hsmar2 ya que se conocía la secuencia consensus.At present, a very small number of active DNA transposons are known, most of which are invertebrates. Only one transposon of mariner family DNA that is active in vertebrates (Sleeping Beauty, from Salmonids) is known, but there is no transposon of mammals, including humans. It is important to highlight that although mariner transposons are relatively autonomous, their activity and efficiency is influenced by the presence / absence of specific cellular factors. Therefore we think that the most efficient transposon to mediate an integrative process in mammals, and especially in human cells, would be through the use of a human transposase, and more specifically Hsmar2 since the consensus sequence was known.
Así pues, la invención se refiere a la generación de Hsmar2, un transposón de ADN humano activo y la posterior demostración y caracterización de su actividad en células humanas.Thus, the invention relates to the generation of Hsmar2 , an active human DNA transposon and the subsequent demonstration and characterization of its activity in human cells.
Figura 1Figure one
Muestra los esquemas de los plásmidos utilizados una vez se han separado la transposasa Hsmar2 de las secuencias ITR que la flanquean. En la, el plásmido pCMV-Hsmar2, el gen Hsmar2 está bajo la acción del promotor viral constitutivo fuerte (CMV) del citomegalovirus y como secuencia de poliadenilación tiene la señal PolyA del virus SV40. En 1b, el plásmido pITR-gen marcador, contiene un cassette de expresión eucariota que contiene un gen marcador (GFP, Bgal, Neo^{R}, etc) flanqueado por las secuencias ITR reconocidas por Hsmar2.It shows the schemes of the plasmids used once the Hsmar2 transposase has been separated from the flanking ITR sequences. In the plasmid pCMV-Hsmar2 , the Hsmar2 gene is under the action of the strong constitutive viral promoter (CMV) of the cytomegalovirus and as a polyadenylation sequence has the PolyA signal of the SV40 virus. In 1b, the plasmid pITR-marker gene contains a eukaryotic expression cassette containing a marker gene (GFP, Bgal, NeoR, etc.) flanked by the ITR sequences recognized by Hsmar2 .
Figura 2Figure 2
Muestra como la transposasa Hsmar2 es activa en células humanas y que el mayor número de clones se obtiene cuando se transfectan células HeLa conjuntamente con los plásmidos que contienen la transposasa (pCMV-Hsmar2) y el transposón (pITR-Neo).It shows how the Hsmar2 transposase is active in human cells and that the greatest number of clones is obtained when HeLa cells are transfected together with the plasmids containing the transposase ( pCMV-Hsmar2 ) and the transposon (pITR-Neo).
Figura 3Figure 3
Muestra que para que la transposición ocurra, la transposasa debe ser activa, ya que cuando se utiliza una transposasa no activa (Hsmar2-AF), la transposición no ocurre. Asimismo, demuestra que a una cantidad fija de transposasa Hsmar2 activa, a mayor cantidad de transposones ITR-Neo, mayor número de eventos de transposición.It shows that for transposition to occur, the transposase must be active, since when a non-active transposase ( Hsmar2 -AF) is used, transposition does not occur. It also demonstrates that at a fixed amount of active Hsmar2 transposase, the greater the number of ITR-Neo transposons, the greater the number of transposition events.
Figura 4Figure 4
Muestra que existe un efecto dosis:respuesta y que con una cantidad fija de de transposones ITR-Neo, a mayor cantidad de transposasa Hsmar2 activa, mayor número de eventos de transposición.It shows that there is a dose: response effect and that with a fixed amount of ITR-Neo transposons, the greater the amount of active Hsmar2 transposase, the greater the number of transposition events.
De izquierda a derecha se observan tres grupos:From left to right there are three groups:
- 1.one.
- Grupo control.Group control.
- 2.2.
- Grupo tratado con 0,4 \mug de pTR-neo:Group treated with 0.4 µg of pTR-neo:
- \circ Columna negra: control.\ circ Black column: control.
- \circ Columna con rayas verticales: 0,1 \mug de Hsmar2.\ circ Column with vertical stripes: 0.1 \ mug of Hsmar2 .
- \circ Columna con puntos: 1,5 \mug de Hsmar2.\ circ Column with points: 1.5 \ mug of Hsmar2 .
- \circ Columna con rayas oblicuas: 4,5 \mug de Hsmar2.\ circ Column with oblique stripes: 4,5 \ mug of Hsmar2 .
\newpage\ newpage
- 3.3.
- Grupo tratado con 4,0 \mug de pTR-neo:Group treated with 4.0 µg of pTR-neo:
- \circ Columna negra:control.\ circ Black column: control.
- \circ Columna con rayas verticales: 0,1 \mug de Hsmar2.\ circ Column with vertical stripes: 0.1 \ mug of Hsmar2 .
- \circ Columna con puntos: 1,5 \mug de Hsmar2.\ circ Column with points: 1.5 \ mug of Hsmar2 .
- \circ Columna con rayas oblicuas: 4,5 \mug de Hsmar2.\ circ Column with oblique stripes: 4,5 \ mug of Hsmar2 .
Figura 5Figure 5
Muestra como la interacción Hsmar2: ITR es específica. Así, en 5a, transfectando células con la transposasa Hsmar2 solo se observa transposición cuando los ITR son los de Hsmar2, pero no cuando son de otro elemento mariner como Seleeping Beauty. En 5b, transfectando células con el gen Neo^{R} flanqueado por los ITR de Hsmar2 solo se observa transposición cuando la transposasa es Hsmar2, pero no cuando es Sleeping Beauty.It shows how the Hsmar2 : ITR interaction is specific. Thus, in 5a, transfecting cells with the Hsmar2 transposase transposition is only observed when the ITRs are those of Hsmar2 , but not when they are from another mariner element such as Seleeping Beauty. In 5b, transfecting cells with the Neo R gene flanked by the Hsmar2 ITRs only transposition is observed when the transposase is Hsmar2 , but not when it is Sleeping Beauty.
Figura 6Figure 6
Muestra el esquema del plásmido que contiene la transposasa quimera GFP:Hsmar2 que permite estudios de localización intracelular. La transposasa quimera GFP:Hsmar2 está bajo la acción del promotor viral constitutivo fuerte (CMV) del citomegalovirus y como secuencia de poliadenilación tiene la señal PolyA del virus SV40.It shows the scheme of the plasmid containing the GFP chimera transposase: Hsmar2 that allows intracellular localization studies. The GFP chimeric transposase: Hsmar2 is under the action of the strong constitutive viral promoter (CMV) of the cytomegalovirus and as a polyadenylation sequence has the PolyA signal of the SV40 virus.
Figura 7Figure 7
Muestra como las transposasa quimera GFP:Hsmar2 es producida en células humanas y como su tamaño es el esperado, y mayor que el de la proteína GFP de la que deriva. Para ello se extrajo proteína total de células HEK-293 transfectadas con GFP o con GFP:Hsmar2, se corrió en un gel de acrilamida y se detectó GFP mediante la utilización de un anticuerpo anti-GFP.It shows how the GFP chimera transposase: Hsmar2 is produced in human cells and how its size is as expected, and larger than that of the GFP protein from which it is derived. For this purpose, total protein was extracted from HEK-293 cells transfected with GFP or with GFP: Hsmar2 , ran on an acrylamide gel and GFP was detected by using an anti-GFP antibody.
De izquierda a derecha se presentan las siguientes columnas:From left to right, the following columns:
- 1.one.
- Células 293 QB transfectadas con pEGFP-C1.293 QB cells transfected with pEGFP-C1.
- 2.2.
- Marcador.Marker.
- 3.3.
- Células 293 QB transfectadas con Hsmar2-GFP.293 QB cells transfected with Hsmar2 -GFP.
Figura 8Figure 8
Muestra la localización intracelular de Hsmar2 en células HeLa y en células HEK-293. Las células fueron analizadas por microscopia de fluorescencia 24 después de la transfección. En células HeLa la localización de Hsmar2 es nuclear, mientras que en células HEK-293 es citoplasmática.It shows the intracellular localization of Hsmar2 in HeLa cells and in HEK-293 cells. The cells were analyzed by fluorescence microscopy 24 after transfection. In HeLa cells the location of Hsmar2 is nuclear, while in HEK-293 cells it is cytoplasmic.
Figura 9Figure 9
A)TO)
\ding{117} pHsmar2-GFP 24 horas.\ ding {117} p Hsmar2 -GFP 24 hours.
\ding{110} pCMV-GFP 24 horas.\ ding {110} pCMV-GFP 24 hours.
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
B)B)
\ding{117} pHsmar2-GFP 24 horas.\ ding {117} p Hsmar2 -GFP 24 hours.
\ding{110} pCMV-GFP 24 horas.\ ding {110} pCMV-GFP 24 hours.
X pHsmar2-GFP 72 horas. X p Hsmar2 -GFP 72 hours.
\medbullet pCMV-GFP 72 horas.pCMV-GFP 72 hours.
Muestra como la expresión de Hsmar2 en células HeLa correlaciona con toxicidad a lo largo del tiempo, mientras que esto no pasa en células HEK-293. En un análisis temprano, a las 24 post-transfección, cuando no es aún posible observar toxicidad por microscopia óptica, los niveles de células que expresan GFP son muy similares en células HeLa y células HEK-293. Sin embargo, en un análisis tardío, a las 72 post-transfección, cuando ya es posible observar toxicidad por microscopia óptica, en células HeLa, los niveles de células GFP-positivas son mucho menores que a las 24 horas, indicando que las células que expresaban GFP:Hsmar2 han muerto. Por el contrario, en células HEK-293 los niveles de células GFP-positivas no solo disminuyen sino que aumentan con respecto a las 24 horas. En el eje se abscisas se muestra los \mug de GFP plasmídico expresado.It shows how the expression of Hsmar2 in HeLa cells correlates with toxicity over time, while this does not happen in HEK-293 cells. In an early analysis, at 24 post-transfection, when it is not yet possible to observe toxicity by optical microscopy, the levels of GFP expressing cells are very similar in HeLa cells and HEK-293 cells. However, in a late analysis, at 72 post-transfection, when it is already possible to observe toxicity by optical microscopy, in HeLa cells, the levels of GFP-positive cells are much lower than at 24 hours, indicating that the cells that They expressed GFP: Hsmar2 have died. In contrast, in HEK-293 cells the levels of GFP-positive cells not only decrease but also increase with respect to 24 hours. On the axis abscissa is shown? Of expressed plasmid GFP.
Inicialmente partimos de la secuencia consensus de Hsmar2 (número de acceso=U49974) y analizamos la base de datos de ADN humano de GenEmbl mediante el programa BLATN 2.0al9MO-WashU. Después de un primer análisis, las secuencias repetidas y las de menos de 600 nucleótidos fueron eliminadas. Se seleccionaron 71 secuencias con una alta homología con U49974 y que contuvieran al menos una de las secuencias ITR. Para el análisis de alineamiento de las secuencias la profundidad mínima fue de 16 y la máxima de 41 y se utilizó la regla de la Mayoría "Alignment by Mayority Rule". Dicha regla se basa en que para cada posición se escoge el nucleótido que esté más representado en cada una de las secuencias estudiadas y alineadas. En nuestro primer análisis detectamos discrepancias entre nuestra secuencia consensus y la secuencia consensus de Hsmar2. Estas diferencias se debían principalmente a cambios en el dinucleótido CG a dinucleótidos CA o TG. In vivo, la causa principal de estos cambios es debido a la hipermutabilidad del dinucleótido CG causada por su metilación. Así, se decidió reintroducir el dinucleótido CG en todos los casos de discrepancia entre nuestra secuencia consensus y la secuencia consensus para Hsmar2 reportada por Roberston (U49974). Es necesario destacar que la mayoría, pero no todos los cambios reintroducidos coincidían con la secuencia consensus U49974 de Hsmar2. En particular los cambios reintroducidos fueron en las posiciones:Initially we start from the consensus sequence of Hsmar2 (access number = U49974) and analyze the GenEmbl human DNA database using the BLATN 2.0al9MO-WashU program. After a first analysis, the repeated sequences and those of less than 600 nucleotides were removed. 71 sequences with high homology to U49974 and containing at least one of the ITR sequences were selected. For the sequence alignment analysis the minimum depth was 16 and the maximum was 41 and the Majority Rule "Alignment by Majority Rule" was used. This rule is based on the fact that for each position the nucleotide that is most represented in each of the sequences studied and aligned is chosen. In our first analysis we detected discrepancies between our consensus sequence and the consensus sequence of Hsmar2 . These differences were mainly due to changes in the CG dinucleotide to CA or TG dinucleotides. In vivo , the main cause of these changes is due to the hypermutability of the CG dinucleotide caused by its methylation. Thus, it was decided to reintroduce the CG dinucleotide in all cases of discrepancy between our consensus sequence and the consensus sequence for Hsmar2 reported by Roberston (U49974). It is necessary to emphasize that the majority, but not all the reintroduced changes coincided with the consensus sequence U49974 of Hsmar2 . In particular, the reintroduced changes were in the positions:
32/33; 89/90; 207/208; 327/328; 342/343;
347/348; 366/367; 371/372; 395/396; 417/418; 438/439;
441/442;
540/541; 556/557; 561/562; 642/643; 667/668;
683/684; 754/755; 759/760; 841/842; 903/904; 907/908; 941/942;
987/988; 996/997; 1191/119232/33; 89/90; 207/208; 327/328; 342/343; 347/348; 366/367; 371/372; 395/396; 417/418; 438/439; 441/442;
540/541; 556/557; 561/562; 642/643; 667/668; 683/684; 754/755; 759/760; 841/842; 903/904; 907/908; 941/942; 987/988; 996/997; 1191/1192
Otras discrepancias observadas con respecto a U49974 fueron:Other discrepancies observed with respect to U49974 were:
- --
- Cambio de Citosina a Timina en la posición 259. Ello implica un cambio de Treonina-26 a Isoleucina-26Change from Cytosine to Timina at position 259. This implies a change of Threonine-26 to Isoleucine-26
- --
- Inserción de una Timina en la posición 1277, en el ITR-3'.Insertion of a thymine in position 1277, in ITR-3 '.
- --
- Cambio de Citosina a Timina en la posición 1280 de nuestra secuencia consensus (o la posición 1281 de U49974), en el ITR-3'.Change from cytosine to thymine at position 1280 of our sequence consensus (or position 1281 of U49974), in the ITR-3 '.
Así, después del análisis confirmamos que el transposón Hsmar2 tiene 1301 pb SEQ ID NO: 1, en la cual, leyendo las posiciones nucleotídicas en dirección 5' \rightarrow 3' se pueden observar diferentes segmentos dentro de la secuencia:Thus, after the analysis we confirm that the Hsmar2 transposon has 1301 bp SEQ ID NO: 1, in which, by reading the nucleotide positions in the 5 '? 3' direction, different segments can be observed within the sequence:
- \ding{51}\ ding {51}
- 1-31: ITRs.1-31: ITRs
- \ding{51}\ ding {51}
- 32-181: región no codificante.32-181: region non-coding
- \ding{51}\ ding {51}
- 182-184: codon start.182-184: codon start
- \ding{51}\ ding {51}
- 1235-1237: codon stop.1235-1237: stop codon.
- \ding{51}\ ding {51}
- 1238-1269: región no codificante.1238-1269: non-coding region.
- \ding{51}\ ding {51}
- 1270-1301:ITRs1270-1301: ITRs
- \ding{51}\ ding {51}
- 258, 1276 y 1280: cambios respecto a la secuencia consenso U49974, de ellos, C258T esta en la región codificante e implica el cambio de Treonina-26 a Isoleucina-26, mientras que los otros dos están dentro de la secuencia ITR-3': el primero es la inserción de una Timina en la posición 1276, y el segundo un cambio de Citosina a Timina en la posición 1280 de nuestra secuencia consensus (o la posición 1281 de U49974).258, 1276 and 1280: changes regarding the consensus sequence U49974, of them, C258T is in the coding region and involves changing Threonine-26 to Isoleucine-26, while the other two are within the sequence ITR-3 ': the first is the insertion of a thymine in position 1276, and the second a change from Cytosine to Timina in the position 1280 of our consensus sequence (or position 1281 of U49974).
Dicha secuencia presenta una pauta de lectura abierta de 1052 pb que codifica los 351 aminoácidos de la transposasa, y contiene el dominio acídico AspAsp(_{34/35})Asp característico de los transposones mariner en las posiciones (Asp_{160}Asp_{254}Asp_{289}) (SEQ ID NO: 2). Asimismo, determinamos que los dos elementos ITR flanqueantes tienen una longitud de 31 pb, y que entre el ITR-5' y el ATG (codon START) de la transposasa hay una región no codificante de 150 pb que posiblemente conformen el promotor que dirige la expresión de Hsmar2; y que entre el codon STOP y el ITR-3' hay una región no codificante de 32 pb que contiene la señal de poliadenilación.This sequence has an open reading pattern of 1052 bp that encodes the 351 amino acids of the transposase, and contains the acidic domain AspAsp (34/35) Asp characteristic of mariner transposons at positions (Asp_ 160 Asp_ { 254} Asp_ {289}) (SEQ ID NO: 2). Likewise, we determine that the two flanking ITR elements have a length of 31 bp, and that between the ITR-5 'and the ATG (START codon) of the transposase there is a non-coding region of 150 bp that possibly forms the promoter that directs the Hsmar2 expression; and that between the STOP codon and the ITR-3 'there is a non-coding region of 32 bp containing the polyadenylation signal.
Finalmente analizamos utilizando la regla de la Mayoría las secuencias que flanqueaban las copias de Hsmar2 presentes en el genoma humano. Así, detectamos el clásico dinucleótido TA justo flanqueando por el exterior a las secuencias ITR, pero además, también pudimos detectar una secuencia consensus de las zonas flanqueantes de Hsmar2, que podría aportar indicios de una secuencia más específica de la zona de inserción. Los números indican la frecuencia en que se encontraban para esa posición. Esta secuencia consensus es:Finally, we analyzed using the Majority rule the sequences that flanked the copies of Hsmar2 present in the human genome. Thus, we detected the classic TA dinucleotide just flanking outside the ITR sequences, but in addition, we could also detect a consensus sequence of the flanking areas of Hsmar2 , which could provide indications of a more specific sequence of the insertion zone. The numbers indicate how often they were for that position. This consensus sequence is:
A_{18/28}(T/A)_{22/28}A_{19/28}T_{24/28}A_{20/28}- -5'-Transposón-3'- -T_{26/29}A_{22/29}T_{18/29}(A/T)_{18/29}T_{21/29}A_ {18/28} (T / A) 22/28} A_ {19/28} T_ {24/28} A_ {20/28} - -5'-Transposon-3'- -T_ {26/29} A_ {22/29} T_ {18/29} (A / T) 18/29} T_ {21/29}
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Asimismo, el análisis de una zona de 30 nucleótidos de cada secuencia flanqueante (65% de AT), muestra que el Hsmar2 se insertaba preferentemente en zonas ricas en el dinucleótido AT.Also, the analysis of an area of 30 nucleotides of each flanking sequence (65% AT), shows that Hsmar2 was preferably inserted in rich areas in the AT dinucleotide.
Debido a que la secuencia consensus no se encuentra dentro del genoma humano ya que todos los transposones identificados contenían multiples mutaciones, decidimos generar de novo la secuencia de Hsmar2 mediante la técnica de PCR recursiva, utilizando un conjunto de 64 oligonucleótidos (32 pares) de 42 bases cada uno. Dichos oligonucleótidos corresponden a las secuencias SEQ ID NO: 2 - SEQ ID NO: 65. Las mutaciones introducidas durante el proceso fueron eliminadas por mutagénesis dirigida hasta obtener la secuencia consensus del transposón Hsmar2. Posteriormente, la región codificante de la transposasa fue escindida y donada en un cassette de expresión con el promotor CMV (Figura 1a), mientras que las secuencias ITR se donaron en un plásmido diferente flanqueando distintos genes marcadores (\beta-Gal, GFP, NeoR) (Figura 1b). De esta manera y para permitir el análisis de la actividad de Hsmar2 separamos en dos plásmidos distintos la transposasa del transposón.Because the consensus sequence is not within the human genome since all the identified transposons contained multiple mutations, we decided to de novo generate the Hsmar2 sequence using the recursive PCR technique, using a set of 64 oligonucleotides (32 pairs) of 42 bases each. Said oligonucleotides correspond to the sequences SEQ ID NO: 2 - SEQ ID NO: 65. The mutations introduced during the process were eliminated by directed mutagenesis until obtaining the consensus sequence of the Hsmar2 transposon. Subsequently, the transposase coding region was cleaved and donated in an expression cassette with the CMV promoter (Figure 1a), while the ITR sequences were donated in a different plasmid flanking different marker genes (β-Gal, GFP, NeoR ) (Figure 1b). In this way and to allow the analysis of the activity of Hsmar2 we separate the transposase from the transposon into two different plasmids.
Esta separación nos permite diseñar experimentos para confirmar la actividad de la transposasa Hsmar2 en células humanas. Así, transducimos células humanas (células HeLa) con un plásmido que contenía las secuencias TR de Hsmar2 que flanquean un cassette de expresión de la resistencia a la neomicina (plásmido TR-Neo), conjuntamente con un plásmido que contenía la transposasa Hsmar2 con el promotor CMV (plásmido CMV-Hsmar2). Como controles, se utilizaron plásmidos con el cassette de neomicina sin estar flanqueado por secuencias TR (plásmido pCi-Neo), y un plásmido con el gen reportero \betaGal bajo la acción del promotor CMV (plásmido pCMV-\betaGal). En el caso de ocurra una transposición, esta solo debería pasar cuando ambos elementos (transposasa y secuencias TR) estén presentes en la célula al mismo tiempo. Tal como se puede observar en la Figura 2, el mayor número de colonias resistentes a la neomicina se da en la condición esperada: plásmido CMV-Hsmar2 + plásmido TR-Neo. Ello indica que la transposasa Hsmar2 es activa y que media específicamente la integración del cassette de resistencia a la neomicina cuando este está flanqueado por las secuencias ITR.This separation allows us to design experiments to confirm the activity of the Hsmar2 transposase in human cells. Thus, we transduced human cells (HeLa cells) with a plasmid containing the Hsmar2 TR sequences flanking a neomycin resistance expression cassette (TR-Neo plasmid), in conjunction with a plasmid containing the Hsmar2 transposase with the promoter CMV (CMV plasmid Hsmar2). As controls, plasmids with the neomycin cassette were used without being flanked by TR sequences (plasmid pCi-Neo), and a plasmid with the βGal reporter gene under the action of the CMV promoter (plasmid pCMV-βGal). In the case of a transposition, this should only happen when both elements (transposase and TR sequences) are present in the cell at the same time. As can be seen in Figure 2, the largest number of neomycin resistant colonies occurs in the expected condition: CMV- Hsmar2 plasmid + TR-Neo plasmid. This indicates that the Hsmar2 transposase is active and that it specifically mediates the integration of the neomycin resistance cassette when it is flanked by the ITR sequences.
Además, tal y como se puede observar en la Figura 3, para que la transposición ocurra es imprescindible que la transposasa Hsmar2 suministrada in trans sea una transposasa activa. Para demostrarlo generamos un nuevo plásmido control (plásmido Hsmar2-AF) que contenía un transposón completo amplificado a partir del locus AF107258 (21q22.1) con una homología de secuencia del 94% con la Hsmar2 consensus, pero con múltiples mutaciones inactivantes. Así, únicamente se observa transposición del transposón TR-Neo cuando la transposasa Hsmar2 activa está presente.In addition, as can be seen in Figure 3, for transposition to occur it is imperative that the Hsmar2 transposase delivered in trans be an active transposase. To demonstrate this, we generated a new control plasmid (plasmid Hsmar2 -AF) that contained a complete transposon amplified from the locus AF107258 (21q22.1) with a sequence homology of 94% with the Hsmar2 consensus, but with multiple inactivating mutations. Thus, only transposition of the TR-Neo transposon is observed when the active Hsmar2 transposase is present.
Seguidamente analizamos si la actividad de Hsmar2 era dependiente de dosis y correlacionaba con la cantidad de transposasa en la célula. Para ello hicimos un experimento similar al anterior, pero ahora variando tanto la cantidad transducida del plásmido TR-Neo como del plásmido CMV-Hsmar2. Así, a más cantidad de transposasa (CMV-Hsmar2), y a mayor cantidad de transposones (TR-Neo) se detecta un mayor número de clones (Figura 4), lo que quiere decir que no sólo los dos elementos son necesarios, sino que el nivel de transposición depende tanto de la su cantidad absoluta tanto de la transposasa como del transposón, así como de su proporción relativa.Next, we analyzed whether the activity of Hsmar2 was dose dependent and correlated with the amount of transposase in the cell. For this we did an experiment similar to the previous one, but now varying both the transduced amount of the TR-Neo plasmid and the CMV- Hsmar2 plasmid. Thus, the more transposase (CMV- Hsmar2 ), and the greater the number of transposons (TR-Neo), a greater number of clones is detected (Figure 4), which means that not only the two elements are necessary, but The level of transposition depends on both its absolute amount of both the transposase and the transposon, as well as its relative proportion.
Otro punto importante es determinar cuan específica es la interacción transposasa:transposón, es decir entre interacción entre la transposasa y las secuencias TR. Para ello, se analizó la capacidad de transposición mediada por Hsmar2, de un cassette de resistencia a la neomicina que estaba flanqueado bien por secuencias ITR de Hsmar2 o por secuencias ITR de Sleeping Beauty (otra transposasa mariner) (Figura 5a). Asimismo, se determinó si las secuencias ITR de Hsmar2 podían ser reconocidas por otras transposasas mariner (Sleeping Beauty) (Figura 5b). Tal como se puede observar, únicamente cuando coincidían la transposasa Hsmar2 con las ITR de Hsmar2 se observaba transposición, indicando que la interacción es muy específica y que no hay "promiscuidad" entre diferentes transposasas. Evidentemente, ello tiene importantes consecuencias a nivel de bioseguridad ya que la expresión de la transposasa Hsmar2 solo permite transponer secuencias de su transposón (flanqueadas por sus ITR), pero no las de otros transposones que puedan estar durmientes o inactivos en el genoma del organismo donde se expresa.Another important point is to determine how specific the transposase: transposon interaction is, that is, between the interaction between the transposase and the TR sequences. For this, the transposition capacity mediated by Hsmar2 , of a neomycin resistance cassette that was flanked either by ITR sequences of Hsmar2 or by ITR sequences of Sleeping Beauty (another mariner transposase) was analyzed (Figure 5a). Likewise, it was determined if the ITR sequences of Hsmar2 could be recognized by other mariner transposases (Sleeping Beauty) (Figure 5b). As it can be seen, only when matched with the transposase Hsmar2 ITRs Hsmar2 transposition was observed, indicating that the interaction is very specific and that no "promiscuous" between different transposase. Obviously, this has important consequences at the biosafety level since the expression of the Hsmar2 transposase only allows transposon sequences (flanked by its ITRs), but not those of other transposons that may be dormant or inactive in the genome of the organism where It is expressed.
Sin embargo, en experimentos con células humanas
HeLa observamos que la sobreexpresión de Hsmar2 provocaba la
muerte celular entre las 24 y las 48 horas posteriores a la
transducción. Estos resultados coinciden con lo reportado para otras
transposasas mariner. Así, parece que la sobreexpresión y posterior
entrada en el núcleo celular induce la generación de cortes o
"nicks" en el genoma de la célula huésped. Si el número de
cortes es suficientemente elevado la célula entra en apoptosis. Sin
embargo, este fenómeno no parece universal porque cuando se
repitieron los experimentos en la línea celular humana 293, esta
citotoxicidad asociada a la sobreexpresión no era observada. Para
determinar las posible causas de la citotoxicidad se generó una
proteína de fusión entre GFP (Green Fluorescent protein) y
Hsmar2 (Figura 6) de manera que mantuviese su actividad, pero
nos fuera posible identificar su localización celular. La actividad
de este constructo fue confirmada repitiendo los experimentos
anteriores. Asimismo, la producción de la proteína quimérica fue
confirmada por Western-Blot utilizando un anticuerpo
contra GFP. Tal como se puede observar en la Figura 7, la proteína
quimera GFP-Hsmar2 tiene un mayor tamaño que la proteína GFP
de la que
deriva.However, in experiments with human HeLa cells we observed that the overexpression of Hsmar2 caused cell death between 24 and 48 hours after transduction. These results coincide with what was reported for other mariner transposases. Thus, it seems that overexpression and subsequent entry into the cell nucleus induces the generation of cuts or "nicks" in the genome of the host cell. If the number of cuts is sufficiently high the cell enters apoptosis. However, this phenomenon does not seem universal because when the experiments were repeated on the human cell line 293, this cytotoxicity associated with overexpression was not observed. To determine the possible causes of cytotoxicity, a fusion protein was generated between GFP (Green Fluorescent protein) and Hsmar2 (Figure 6) so as to maintain its activity, but we could identify its cellular location. The activity of this construct was confirmed by repeating the previous experiments. Also, the production of the chimeric protein was confirmed by Western-Blot using an antibody against GFP. As can be seen in Figure 7, the GFP-Hsmar2 chimera protein is larger than the GFP protein of which
drift.
La transducción de la transposasa quimérica GFP-Hsmar2 en células HeLa y en células HEK-293 permite observar como en las células HeLa su localización es nuclear, mientras que en las células 293 su localización es mayoritariamente citoplasmática (Figura 8). Es decir, permite correlacionar de manera directa entre citotoxicidad y localización celular: cuando la transposasa se localiza en el núcleo celular (como en las células HeLa) es capaz de generar cortes en el genoma lo que a partir de un cierto nivel inducirá a la célula a entrar en apoptosis. Por el contrario, cuando la transposasa está localizada en el citoplasma (células HEK-293), donde no es capaz de generar cortes en el genoma y por lo tanto la célula no entrará en apoptosis.The transduction of the GFP-Hsmar2 chimeric transposase in HeLa cells and in HEK-293 cells makes it possible to observe how in HeLa cells its location is nuclear, while in 293 cells its location is mostly cytoplasmic (Figure 8). That is, it allows a direct correlation between cytotoxicity and cellular localization: when the transposase is located in the cell nucleus (as in HeLa cells), it is capable of generating cuts in the genome, which will induce the cell from a certain level. to enter apoptosis. On the contrary, when the transposase is located in the cytoplasm (HEK-293 cells), where it is not able to generate cuts in the genome and therefore the cell will not enter apoptosis.
Finalmente, se determinó si el grado de toxicidad estaba asociado a niveles elevados de la transposasa en núcleo o bien tan solo a su presencia en él aunque fuese a niveles bajos. Para ello, se transdujeron células HeLa con distintas cantidades del plásmido GFP-Hsmar2 y del plásmido control pCMV-GFP, y se analizó el porcentaje de células transducidas vivas en cada condición a lo largo del tiempo mediante FACS (Fluorescence Activated Cell Sorting). Como se puede observar en la Figura 9, el porcentaje de células vivas que expresan la proteína GFP cuando son transducidas con el plásmido control pCMV-GFP aumenta con el tiempo, mientras que el porcentaje de células vivas que expresan la proteína GFP cuando son transducidas con el plásmido pHsmar2-GFP disminuye con el tiempo, seguramente a una toxicidad mediada por Hsmar2.Finally, it was determined whether the degree of toxicity was associated with elevated levels of the transposase in the nucleus or only with its presence in it although it was at low levels. For this, HeLa cells were transduced with different amounts of the GFP- Hsmar2 plasmid and the control plasmid pCMV-GFP, and the percentage of live transduced cells in each condition over time was analyzed by FACS (Fluorescence Activated Cell Sorting). As can be seen in Figure 9, the percentage of live cells expressing the GFP protein when transduced with the control plasmid pCMV-GFP increases over time, while the percentage of live cells expressing the GFP protein when transduced with the plasmid pHsmar2 -GFP decreases over time, probably at a toxicity mediated by Hsmar2 .
Así, en un primer aspecto la presente invención se refiere al transposón Hsmar2, caracterizado por ser activo en células de mamífero, cuya estructura comprende una secuencia de ADN susceptible de ser transferida génicamente y dos secuencias ITR flanqueantes de dicha secuencia de ADN.Thus, in a first aspect the present invention relates to the Hsmar2 transposon, characterized by being active in mammalian cells, whose structure comprises a DNA sequence capable of being genetically transferred and two flanking ITR sequences of said DNA sequence.
En un aspecto preferido la presente invención se refiere al transposón Hsmar2 caracterizado por ser activo en células de mamífero, cuya estructura comprende una secuencia de ADN susceptible de ser transferida génicamente y dos secuencias ITR flanqueantes de dicha secuencia de ADN, definido por la secuencia SEQ ID NO: 1 al corresponderse, la secuencia de ADN susceptible de ser transferida génicamente y flanqueada por las dos secuencias ITR, con la secuencia codificante de la enzima transposasa.In a preferred aspect the present invention relates to the Hsmar2 transposon characterized by being active in mammalian cells, whose structure comprises a DNA sequence capable of being genetically transferred and two flanking ITR sequences of said DNA sequence, defined by the sequence SEQ ID NO: 1 when applicable, the DNA sequence that can be genetically transferred and flanked by the two ITR sequences, with the transposase enzyme coding sequence.
Tal y como se utiliza en la invención, el término "transposasa" se refiere a la proteína que realiza la reacción de trasposición de un fragmento de ADN a un genoma receptor.As used in the invention, the term "transposase" refers to the protein that performs the transposition reaction of a DNA fragment to a genome receiver.
Por otro lado, el término "transposón" se refiere a un fragmento de ADN formado por la secuencia nucleotídica de un gen (pudiendo ser codificante o no), un promotor que dirige la expresión de dicho gen (en caso de que la secuencia nucleotídica sea codificante), y dos secuencias ITR terminales o flanqueantes de dicha secuencia nucleotídica. Por lo tanto, la secuencia nucleotídica (comprendida entre las secuencias ITR flanqueantes) del transposón puede ser la de cualquier gen susceptible de ser transferida e insertada en el genoma de la célula receptora (en la presente invención es la secuencia del gen de resistencia a Neomicina), suministrándose en este caso la Transposasa en trans mediante un segundo vector (plásmido) bajo la acción de un promotor que sería el encargado de dirigir la expresión del gen de la transposasa. Por lo tanto, en este caso el sistema estaría formado por dos componentes tal y como se detalla en la figura 1.On the other hand, the term "transposon" is refers to a DNA fragment formed by the nucleotide sequence of a gene (can be coding or not), a promoter that directs the expression of said gene (in case the nucleotide sequence be coding), and two terminal or flanking ITR sequences of said nucleotide sequence. Therefore the sequence nucleotide (comprised between flanking ITR sequences) of the transposon can be that of any gene likely to be transferred and inserted into the genome of the recipient cell (in the present invention is the sequence of the resistance gene to Neomycin), the Transposase being supplied in this case in trans by a second vector (plasmid) under the action of a promoter that would be responsible for directing the expression of the gene of the transposase Therefore, in this case the system would be formed by two components as detailed in figure 1.
De forma particular la secuencia nucleotídica (comprendida entre las secuencias ITR flanqueantes) del transposón puede corresponderse con la secuencia codificante de la transposasa, con lo cual no sería necesario que ésta fuera administrada en trans. En este caso el transposón se correspondería con la SEQ ID NO: 1.In particular the nucleotide sequence (comprised between flanking ITR sequences) of the transposon may correspond to the coding sequence of the transposase, so it would not be necessary for it to be administered in trans. In this case the transposon would correspond with SEQ ID NO: 1.
Tal y como se utiliza en la presente invención el término "transferencia génica" se refiere a un proceso por el cual las secuencias de material genético (ADN o ARN) se introducen en la célula hospedadora. Dicha introducción está mediada por vectores que protegen el material genético y facilitan su unión y entrada en la célula hospedadora previo transporte al núcleo de la misma. En una realización preferida de la invención la transferencia génica, y por lo tanto la introducción del material genético en las célula hospedadora, se realiza con fines terapéuticos, es decir, se realiza "terapia génica".As used in the present invention the term "gene transfer" refers to a process by which the sequences of genetic material (DNA or RNA) are They enter the host cell. This introduction is mediated by vectors that protect the genetic material and facilitate its union and entry into the host cell prior to transport to the nucleus of the same. In a preferred embodiment of the invention the gene transfer, and therefore the introduction of the material genetic in the host cell, is done for purposes therapeutic, that is, "gene therapy" is performed.
En un segundo aspecto la presente invención se refiere al uso del transposón Hsmar2 cuya estructura está comprendida por una secuencia de ADN susceptible de ser transferida génicamente y dos secuencias ITR flanqueantes de dicha secuencia de ADN para la generación de vectores con capacidad de integración en el genoma de una célula hospedadora.In a second aspect the present invention is refers to the use of the Hsmar2 transposon whose structure is comprised of a DNA sequence that can be transferred genetically and two flanking ITR sequences of said sequence of DNA for vector generation with integration capability in the genome of a host cell.
En un aspecto preferido la presente invención se refiere al uso del transposón Hsmar2, cuya estructura definida por la SEQ ID NO: 1, para la generación de vectores con capacidad de integración en el genoma de una célula hospedadora.In a preferred aspect the present invention is refers to the use of the Hsmar2 transposon, whose structure defined by SEQ ID NO: 1, for the generation of vectors capable of Integration into the genome of a host cell.
En otro aspecto preferido la presente invención se refiere al uso del transposón Hsmar2 cuya estructura está comprendida por una secuencia de ADN susceptible de ser transferida génicamente y dos secuencias ITR flanqueantes de dicha secuencia de ADN o definida por la SEQ ID NO: 1, para la generación de vectores con capacidad de integración en el genoma de una célula hospedadora, donde el vector con capacidad de integración generado es un Adenovirus.In another preferred aspect the present invention refers to the use of the Hsmar2 transposon whose structure is comprised of a DNA sequence that can be transferred genetically and two flanking ITR sequences of said sequence of DNA or defined by SEQ ID NO: 1, for the generation of vectors with ability to integrate into the genome of a cell host, where the vector with generated integration capacity It is an adenovirus.
En otro aspecto preferido la presente invención se refiere al uso de los vectores que comprenden el transposón Hsmar2 cuya estructura está comprendida por una secuencia de ADN susceptible de ser transferida génicamente y dos secuencias ITR flanqueantes de dicha secuencia, en la elaboración de composiciones destinadas a la realización de transferencia génica.In another preferred aspect the present invention refers to the use of vectors comprising the transposon Hsmar2 whose structure is comprised of a DNA sequence liable to be genetically transferred and two ITR sequences flanks of said sequence, in the elaboration of compositions destined to the realization of gene transfer.
En otro aspecto preferido la presente invención se refiere al uso de los vectores que comprenden el transposón Hsmar2 cuya estructura está definida por la SEQ ID NO: 1, en la elaboración de composiciones destinadas a la realización de transferencia génica.In another preferred aspect the present invention refers to the use of vectors comprising the transposon Hsmar2 whose structure is defined by SEQ ID NO: 1, in the preparation of compositions intended for the realization of gene transfer
En otro aspecto preferido la presente invención se refiere al uso de los vectores que comprenden el transposón Hsmar2 cuya estructura está comprendida por una secuencia de ADN susceptible de ser transferida génicamente y dos secuencias ITR flanqueantes de dicha secuencia o definida por la SEQ ID NO: 1, en la elaboración de composiciones destinadas a la realización de transferencia génica, donde dicha transferencia génica se realiza "in vivo" en mamíferos.In another preferred aspect the present invention relates to the use of vectors comprising the Hsmar2 transposon whose structure is comprised of a DNA sequence capable of being genetically transferred and two ITR sequences flanking said sequence or defined by SEQ ID NO: 1 , in the elaboration of compositions destined to the realization of gene transfer, where said gene transfer is carried out " in vivo " in mammals.
En otro aspecto preferido la presente invención se refiere al uso de los vectores que comprenden el transposón Hsmar2 cuya estructura está comprendida por una secuencia de ADN susceptible de ser transferida génicamente y dos secuencias ITR flanqueantes de dicha secuencia o definida por la SEQ ID NO: 1, en la elaboración de composiciones destinadas a la realización de transferencia génica, donde dicha transferencia génica se realiza "in vivo" en humanos.In another preferred aspect the present invention relates to the use of vectors comprising the Hsmar2 transposon whose structure is comprised of a DNA sequence capable of being genetically transferred and two ITR sequences flanking said sequence or defined by SEQ ID NO: 1 , in the elaboration of compositions destined to the realization of gene transfer, where said gene transfer is carried out " in vivo " in humans.
En otro aspecto preferido la presente invención se refiere al uso de los vectores que comprenden el transposón Hsmar2 cuya estructura está comprendida por una secuencia de ADN susceptible de ser transferida génicamente y dos secuencias ITR flanqueantes de dicha secuencia o definida por la SEQ ID NO: 1, en la elaboración de composiciones destinadas a la realización de transferencia génica, donde dicha transferencia génica se realiza "in vitro" en cultivos de células de mamíferos.In another preferred aspect the present invention relates to the use of vectors comprising the Hsmar2 transposon whose structure is comprised of a DNA sequence capable of being genetically transferred and two ITR sequences flanking said sequence or defined by SEQ ID NO: 1 , in the elaboration of compositions destined to the realization of gene transfer, where said gene transfer is carried out " in vitro " in mammalian cell cultures.
En otro aspecto preferido la presente invención se refiere al uso de los vectores que comprenden el transposón Hsmar2 cuya estructura está comprendida por una secuencia de ADN susceptible de ser transferida génicamente y dos secuencias ITR flanqueantes de dicha secuencia o definida por la SEQ ID NO: 1, en la elaboración de composiciones destinadas a la realización de transferencia génica, donde dicha transferencia génica se realiza "in vitro" en cultivos de células de humanos.In another preferred aspect the present invention relates to the use of vectors comprising the Hsmar2 transposon whose structure is comprised of a DNA sequence capable of being genetically transferred and two ITR sequences flanking said sequence or defined by SEQ ID NO: 1 , in the elaboration of compositions destined to the realization of gene transfer, where said gene transfer is carried out " in vitro " in human cell cultures.
Un tercer aspecto de la presente invención se refiere a un vector que comprende el transposón Hsmar2 cuya estructura está comprendida por una secuencia de ADN susceptible de ser transferida génicamente y dos secuencias ITR flanqueantes de dicha secuencia codificante.A third aspect of the present invention is refers to a vector comprising the transposon Hsmar2 whose structure is comprised of a DNA sequence susceptible to be genetically transferred and two flanking ITR sequences from said coding sequence.
En un aspecto preferido la presente invención se refiere a un vector que comprende el transposón Hsmar2 cuya estructura está definida por la SEQ ID NO: 1.In a preferred aspect the present invention is refers to a vector comprising the transposon Hsmar2 whose structure is defined by SEQ ID NO: 1.
En otro aspecto preferido la presente invención se refiere a un vector que comprende el transposón Hsmar2 cuya estructura está comprendida por una secuencia de ADN susceptible de ser transferida génicamente y dos secuencias ITR flanqueantes de dicha secuencia codificante, o definida por la SEQ ID NO: 1, donde el vector es un Adenovirus.In another preferred aspect the present invention refers to a vector comprising the transposon Hsmar2 whose structure is comprised of a DNA sequence susceptible to be genetically transferred and two flanking ITR sequences from said coding sequence, or defined by SEQ ID NO: 1, where The vector is an adenovirus.
Un cuarto aspecto de la presente invención se refiere a células transferidas génicamente por un vector que comprende el transposón Hsmar2 cuya estructura está comprendida por una secuencia de ADN susceptible de ser transferida génicamente y dos secuencias ITR flanqueantes de dicha secuencia de ADN.A fourth aspect of the present invention is refers to cells genetically transferred by a vector that it comprises the Hsmar2 transposon whose structure is comprised of a DNA sequence capable of being genetically transferred and two flanking ITR sequences of said DNA sequence.
Un aspecto preferido de la presente invención se refiere a células transferidas génicamente por un vector que comprende el transposón Hsmar2 cuya estructura está definida por la SEQ ID NO: 1.A preferred aspect of the present invention is refers to cells genetically transferred by a vector that comprises the transposon Hsmar2 whose structure is defined by the SEQ ID NO: 1.
Un quinto aspecto de la presente invención se refiere al uso de las células transferidas génicamente por un vector que comprende el transposón Hsmar2 cuya estructura está comprendida por una secuencia de ADN susceptible de ser transferida génicamente y dos secuencias ITR flanqueantes de dicha secuencia de ADN, para la elaboración de composiciones farmacéuticas destinadas a ser utilizadas en transferencia génica.A fifth aspect of the present invention is refers to the use of genetically transferred cells by a vector comprising the transposon Hsmar2 whose structure is comprised of a DNA sequence that can be transferred genetically and two flanking ITR sequences of said sequence of DNA, for the preparation of pharmaceutical compositions intended to be used in gene transfer.
Un aspecto preferido de la presente invención se refiere al uso de las células transferidas génicamente por un vector que comprende el transposón Hsmar2 cuya estructura está definida por la SEQ ID NO: 1 para la elaboración de composiciones farmacéuticas destinadas a ser utilizadas en transferencia génica.A preferred aspect of the present invention is refers to the use of genetically transferred cells by a vector comprising the transposon Hsmar2 whose structure is defined by SEQ ID NO: 1 for the preparation of compositions pharmaceuticals intended to be used in transfer gene.
Un sexto aspecto de la presente invención se refiere a composiciones farmacéuticas que contienen vectores que comprenden el transposón Hsmar2 cuya estructura está comprendida por una secuencia de ADN susceptible de ser transferida génicamente y dos secuencias ITR flanqueantes de dicha secuencia de ADN, y vehículos farmacéuticamente aceptables.A sixth aspect of the present invention is refers to pharmaceutical compositions containing vectors that comprise the Hsmar2 transposon whose structure is comprised by a DNA sequence that can be genetically transferred and two flanking ITR sequences of said DNA sequence, and pharmaceutically acceptable vehicles.
Un aspecto preferido de la presente invención se refiere a composiciones farmacéuticas que contienen vectores que comprenden el transposón Hsmar2 cuya estructura está definida por la SEQ ID NO: 1, y vehículos farmacéuticamente aceptables.A preferred aspect of the present invention is refers to pharmaceutical compositions containing vectors that they comprise the transposon Hsmar2 whose structure is defined by SEQ ID NO: 1, and pharmaceutically acceptable vehicles.
Otro aspecto preferido de la presente invención se refiere a composiciones farmacéuticas que contienen células transferidas génicamente por vectores que comprenden el transposón Hsmar2 cuya estructura está comprendida por una secuencia de ADN susceptible de ser transferida génicamente y dos secuencias ITR flanqueantes de dicha secuencia de ADN y vehículos farmacéuticamente aceptables.Another preferred aspect of the present invention refers to pharmaceutical compositions containing cells genetically transferred by vectors comprising the transposon Hsmar2 whose structure is comprised of a DNA sequence liable to be genetically transferred and two ITR sequences flanks of said DNA sequence and vehicles pharmaceutically acceptable.
Otro aspecto preferido de la presente invención se refiere a composiciones farmacéuticas que contienen células transferidas génicamente por vectores que comprenden el transposón Hsmar2 cuya estructura está definida por la SEQ ID NO: 1, y vehículos farmacéuticamente aceptables.Another preferred aspect of the present invention refers to pharmaceutical compositions containing cells genetically transferred by vectors comprising the transposon Hsmar2 whose structure is defined by SEQ ID NO: 1, and pharmaceutically acceptable vehicles.
\newpage\ newpage
Un séptimo aspecto de la presente invención se refiere a un método de transfección celular que consiste en la introducción en una célula hospedadora de un vector que comprende el transposón Hsmar2 cuya estructura está comprendida por una secuencia de ADN susceptible de ser transferida génicamente y dos secuencias ITR flanqueantes de dicha secuencia de ADN, el cual comprende la transfección adicional de la Transposasa en trans, utilizando un segundo vector.A seventh aspect of the present invention is refers to a method of cellular transfection consisting of the introduction into a host cell of a vector comprising the Hsmar2 transposon whose structure is comprised of a DNA sequence capable of being genetically transferred and two flanking ITR sequences of said DNA sequence, which comprises additional transfection of Transposase in trans, Using a second vector.
Un aspecto preferido de la presente invención se refiere a un método de transfección celular que consiste en la introducción en una célula hospedadora de un vector que comprende el transposón Hsmar2 cuya estructura está definida por la SEQ ID NO: 1.A preferred aspect of the present invention is refers to a method of cellular transfection consisting of the introduction into a host cell of a vector comprising Hsmar2 transposon whose structure is defined by SEQ ID NO: one.
Otro aspecto preferido de la presente invención se refiere a método de transfección celular que consiste en la introducción en una célula hospedadora de un vector que comprende el transposón Hsmar2 cuya estructura está comprendida por una secuencia de ADN susceptible de ser transferida génicamente y dos secuencias ITR flanqueantes de dicha secuencia de ADN, el cual comprende la transfección adicional de la Transposasa en trans utilizando un segundo vector, o que consiste en la introducción en una célula hospedadora de un vector que comprende el transposón Hsmar2 cuya estructura está definida por la SEQ ID NO: 1, donde las células transfectadas son de mamífero.Another preferred aspect of the present invention refers to cell transfection method consisting of the introduction into a host cell of a vector comprising the Hsmar2 transposon whose structure is comprised of a DNA sequence capable of being genetically transferred and two flanking ITR sequences of said DNA sequence, which comprises additional transfection of Transposase in trans using a second vector, or consisting of the introduction in a host cell of a vector comprising the transposon Hsmar2 whose structure is defined by SEQ ID NO: 1, where Transfected cells are from mammalian.
Otro aspecto preferido de la presente invención se refiere a método de transfección celular que consiste en la introducción en una célula hospedadora de un vector que comprende el transposón Hsmar2 cuya estructura está comprendida por una secuencia de ADN susceptible de ser transferida génicamente y dos secuencias ITR flanqueantes de dicha secuencia de ADN, el cual comprende la transfección adicional de la Transposasa en trans utilizando un segundo vector, o que consiste en la introducción en una célula hospedadora de un vector que comprende el transposón Hsmar2 cuya estructura está definida por la SEQ ID NO: 1, donde las células transfectadas son humanas.Another preferred aspect of the present invention refers to cell transfection method consisting of the introduction into a host cell of a vector comprising the Hsmar2 transposon whose structure is comprised of a DNA sequence capable of being genetically transferred and two flanking ITR sequences of said DNA sequence, which comprises additional transfection of Transposase in trans using a second vector, or consisting of the introduction in a host cell of a vector comprising the transposon Hsmar2 whose structure is defined by SEQ ID NO: 1, where Transfected cells are human.
Otro aspecto preferido de la presente invención se refiere a método de transfección celular que consiste en la introducción en una célula hospedadora de un vector que comprende el transposón Hsmar2 cuya estructura está comprendida por una secuencia de ADN susceptible de ser transferida génicamente y dos secuencias ITR flanqueantes de dicha secuencia de ADN, el cual comprende la transfección adicional de la Transposasa en trans utilizando un segundo vector, o que consiste en la introducción en una célula hospedadora de un vector que comprende el transposón Hsmar2 cuya estructura está definida por la SEQ ID NO: 1, donde el vector es un Adenovirus.Another preferred aspect of the present invention refers to cell transfection method consisting of the introduction into a host cell of a vector comprising the Hsmar2 transposon whose structure is comprised of a DNA sequence capable of being genetically transferred and two flanking ITR sequences of said DNA sequence, which comprises additional transfection of Transposase in trans using a second vector, or consisting of the introduction in a host cell of a vector comprising the transposon Hsmar2 whose structure is defined by SEQ ID NO: 1, where the Vector is an adenovirus.
La exposición detallada de los ejemplos y de las figuras que siguen, se proporcionan a modo de ilustración y no pretenden ser limitantes de la presente invención.The detailed exposition of the examples and the figures that follow are provided by way of illustration and not They are intended to be limiting of the present invention.
Los siguientes ejemplos describen los procedimientos utilizados tanto para la generación de la transposasa Hsmar2 como analizar su actividad.The following examples describe the procedures used both for the generation of the Hsmar2 transposase and to analyze its activity.
Para llevar a cabo estos ensayos, se utilizaron las metodologías que se describen a continuación:To carry out these tests, they were used The methodologies described below:
Para la obtención de ADN plasmídico en bajas cantidades (5-15 \mug) se realizan minipreparaciones de ADN por lisis alcalina a partir de 3 mL de cultivo. Este método se basa en la lisis de las bacterias a partir de un tampón alcalino, seguido de una precipitación de proteína con acetato potásico y una posterior precipitación del plásmido mediante isopropanol y un lavado final con etanol al 70%. El ARN es eliminado mediante RNAsa A.For obtaining plasmid DNA in casualties quantities (5-15 \ mug) are made DNA minipreparations by alkaline lysis from 3 mL of culture. This method is based on the lysis of the bacteria from of an alkaline buffer, followed by a precipitation of protein with potassium acetate and subsequent plasmid precipitation by isopropanol and a final wash with 70% ethanol. RNA is removed by RNAse A.
- --
- Solución de resuspensión: 50 mM Tris-Cl a pH 8,0; 10 mM EDTA; 100 \mug/mL RNAsaA.Resuspension solution: 50 mM Tris-Cl at pH 8.0; 10 mM EDTA; 100 µg / mL RNAsaA.
- --
- Solución de lisis: 200 mM NaOH; 1% SDS (w/v).Lysis solution: 200 mM NaOH; 1% SDS (w / v).
- --
- Solución de precipitado proteico: 3,0 M Acetato potásico pH 5,5Protein precipitate solution: 3.0 M Potassium acetate pH 5.5
La purificación de ADN en geles de agarosa se realizó mediante el kit GENECLEAN Turbo Kit de Q-BIOgene (Irvine, California, EE.UU.) siguiendo el protocolo que ofrece el fabricante.DNA purification in agarose gels is made using the GENECLEAN Turbo Kit Q-BIOgene (Irvine, California, USA) following the protocol offered by the manufacturer.
Los enzimas utilizados en este trabajo provienen de la marca FERMENTAS y NEW ENGLAND Biolabs. Se han utilizado 5U de digestión para 2 \mug de ADN plasmídico en las condiciones de temperatura y solución establecidas por el fabricante. El tiempo de digestión varía de 1-18 horas en función de la dosis administrada. La ligasa y fosfatasa alcalina se han utilizado en las dosis y tiempos establecidos por el fabricante.The enzymes used in this work come from of the brand FERMENTAS and NEW ENGLAND Biolabs. 5U of digestion for 2 µg of plasmid DNA under the conditions of temperature and solution set by the manufacturer. The time of Digestion varies from 1-18 hours depending on the dose managed. Ligase and alkaline phosphatase have been used in the doses and times established by the manufacturer.
La secuenciación de las secuencias generadas se realizó tanto en el Servicio de Secuenciación de Genethon III (Evry, Francia) como en el Servicio de Secuenciación de la Facultad de Veterinaria de la Universidad Autónoma de Barcelona.The sequencing of the generated sequences is performed both in the Genethon III Sequencing Service (Evry, France) as in the Sequencing Service of the Faculty of Veterinary of the Autonomous University of Barcelona.
Paso 1º:Step 1:
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Los oligonucleótidos arriba citados se encuentran representados por las secuencias SEQ ID NO: 2 - SEQ ID NO: 65.The oligonucleotides mentioned above are are represented by the sequences SEQ ID NO: 2 - SEQ ID NO: 65
Con estos oligos se amplificó una banda de 140 bp mediante 30 ciclos de PCR.With these oligos a band of 140 was amplified bp by 30 cycles of PCR.
1 pmol cada oligonucleótido1 pmol each oligonucleotide
5 \mu1 buffer Taq (sin Mg^{++})5 µ1 Taq buffer (without Mg ++)
1 \mul MgCl_{2} (60 mM)1 µM MgCl2 (60 mM)
0,8 \mul dNTPs (1.25 mM)0.8 µL dNTPs (1.25 mM)
1 unidad Taq Pol + 0,03 \mul Taq Ultma1 unit Taq Pol + 0,03 \ mul Taq Ultma
agua milliQ hasta 50 \mulMilliQ water up to 50 \ mul
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
- Condiciones: Terms:
- 94ºC, 30 sec,94 ° C, 30 sec,
- \quadquad
- 94ºC 30 seg,94 ° C 30 sec,
- \quadquad
- 45ºC + 0,4ºC/ciclo 30 seg,45ºC + 0.4ºC / cycle 30 sec,
- \quadquad
- 72ºC 20 sec + 0,5 seg/ciclo72ºC 20 sec + 0.5 sec / cycle
- \quadquad
- durante 20 ciclos,for 20 cycles,
- \quadquad
- 72ºC, 2 min72 ° C, 2 min
- \quadquad
- 25ºC, 2 min25 ° C, 2 min
\newpage\ newpage
Paso 2º:Step 2:
10 \mul buffer Taq (sin Mg^{++})10 µL buffer Taq (without Mg ++)
2 \mul MgCl_{2} (60 mM)2 µM MgCl 2 (60 mM)
1,6 \mul dNTPs (1.25 mM)1.6 µL dNTPs (1.25 mM)
1 unidad Taq Pol + 0,03 \mul Taq Ultma1 unit Taq Pol + 0,03 \ mul Taq Ultma
6 \mul de cada PCR previa (fragmentos A +B para generar nuevo fragmento JP-16 µl of each previous PCR (fragments A + B to generate new JP-1 fragment
fragmentos C + D + E para generar nuevo fragmento JP-2C + D + E fragments to generate new JP-2 fragment
agua milliQ hasta 100 \mulMilliQ water up to 100 \ mul
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
- Condiciones: Terms:
- 94ºC, 30 sec,94 ° C, 30 sec,
- \quadquad
- 94ºC 30 seg,94 ° C 30 sec,
- \quadquad
- 50ºC + 0,2ºC/ciclo 30 seg,50ºC + 0.2ºC / cycle 30 sec,
- \quadquad
- 72ºC 50 sec72ºC 50 sec
- \quadquad
- durante 15 ciclos,for 15 cycles,
- Añadir 40 pmol de oligos: Add 40 pmol of oligos:
- MC-D-1 + MC-R2-18 para fragmento JP-1MC-D-1 + MC-R2-18 for fragment JP-1
- \quadquad
- MC-D-14 + MC-R2-1 para fragmento JP-2MC-D-14 + MC-R2-1 for fragment JP-2
- \quadquad
- Hacer 15 ciclos másDo 15 more cycles
- \quadquad
- 72ºC, 2 min72 ° C, 2 min
- \quadquad
- 25ºC, 2 min25 ° C, 2 min
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Paso 3º:Step 3:
- --
- Purificación de los fragmentos de PCRPurification of the fragments of PCR
- --
- Clonación en plásmido p123TPlasmid p123T cloning
- --
- Secuenciación de al menos 20 clones diferentes de cada construcción p123T/PJ-1 o p123T/PJ-2Sequencing of at least 20 clones different from each construction p123T / PJ-1 or p123T / PJ-2
- --
- Para cada construcción, se seleccionó el clon con menos mutaciones con respecto a la secuencia esperada.For each construct, the clone with fewer mutations was selected with Regarding the expected sequence.
- --
- Eliminación de las mutaciones no deseadas mediante mutagénesis dirigida utilizando el kit QuickChange Site-Directed Mutagenesis (Stratagene)Elimination of non-mutations desired by directed mutagenesis using the QuickChange kit Site-Directed Mutagenesis (Stratagene)
- --
- Combinación de ambos fragmentos mediante la utilización de la diana interna PstI que es única en ambos constructos (posición 573 de JP) para generar el transposón completo Hsmar2.Combination of both fragments by using the internal PstI target that is unique in both constructs (position 573 of JP) to generate the complete transposon Hsmar2 .
- --
- Secuenciación final para confirmar la ausencia de mutaciones en la construcción generada.Final sequencing to confirm the absence of mutations in the generated construction.
Mediante PCR se amplificó el transposón Hsmar2 inactivo presente en el locus AF1072585 localizado en el 21q22.1. Las condiciones de amplificación fueron:The inactive Hsmar2 transposon present in the locus AF1072585 located at 21q22.1 was amplified by PCR. The amplification conditions were:
100 ng de ADN humano (extraído de células HeLa)100 ng of human DNA (extracted from cells HeLa)
40 pmol cada oligonucleótido40 pmol each oligonucleotide
10 \mul buffer Taq (sin Mg^{++})10 µL buffer Taq (without Mg ++)
5 \mul MgCl_{2} (60 mM)5 µM MgCl 2 (60 mM)
1,6 \mul dNTPs (1.25 mM)1.6 µL dNTPs (1.25 mM)
2 unidades Taq Pol + 0,03 \mul Taq Ultma2 units Taq Pol + 0,03 \ mul Taq Ultma
agua milliQ hasta 100 \mulMilliQ water up to 100 \ mul
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
- Condiciones: Terms:
- 94ºC, 30 sec,94 ° C, 30 sec,
- \quadquad
- 94ºC 30 seg,94 ° C 30 sec,
- \quadquad
- 48ºC 30 seg,48ºC 30 sec,
- \quadquad
- 74ºC 30 sec74ºC 30 sec
- \quadquad
- durante 35 ciclos,for 35 cycles,
- \quadquad
- 72ºC, 2 min72 ° C, 2 min
- \quadquad
- 25ºC, 2 min25 ° C, 2 min
El fragmento de PCR fue donado en el plásmido pCR-TOPO (Invitrogen) siguiendo las instrucciones del fabricante. Posteriormente plásmidos con patrón de restricción positivo fueron secuenciados para confirmar la secuencia del transposón Hsmar2 del locus AF107258.The PCR fragment was donated in plasmid pCR-TOPO (Invitrogen) following the manufacturer's instructions. Subsequently plasmids with positive restriction pattern were sequenced to confirm the Hsmar2 transposon sequence of the AF107258 locus.
Las células (HeLa o HEK-293) se cultivaron en placas de 6 pocillos en medio de cultivo DMEM + 10% FBS. Cuando están a un 80% de confluencia (alrededor de 800.000 células por pocillo) están listas para transfectar. La transfección se hace utilizando PEI (Sigma) a una proporción de 2.25 111 (10 mM) por cada microgramo de ADN, utilizando como norma, 6 \mug de ADN por pocillo. Se dejan formar los complejos durante 30 minutos y luego se añaden a las células durante 4 horas. Después de este tiempo se cambia el medio de las células por medio fresco con DMEM y 10% FBS y se incuban a 37ºC y 5% CO_{2} hasta su análisis.The cells (HeLa or HEK-293) are cultured in 6-well plates in DMEM culture medium + 10% FBS When they are at 80% confluence (around 800,000 cells per well) are ready to transfect. Transfection it is done using PEI (Sigma) at a ratio of 2.25 111 (10 mM) for each microgram of DNA, using 6 µg of DNA as a rule per well. The complexes are allowed to form for 30 minutes and Then they are added to the cells for 4 hours. After this time the medium of the cells is changed to fresh medium with DMEM and 10% FBS and incubated at 37 ° C and 5% CO2 until analysis.
Las células se tripsinizan 48 horas después de la transfección y se cultivan en placas de 10-cm en presencia de medio DMEM con 10% FBS y neomicina (600 \mug/ml). Cada dos días se añade medio fresco DMEM con 10% FBS y neomicina (600 \mug/ml), hasta que los clones individuales son claramente visibles.The cells are trypsinized 48 hours after the transfection and are grown in 10-cm plates in presence of DMEM medium with 10% FBS and neomycin (600 µg / ml). DMEM fresh medium with 10% FBS and neomycin is added every two days (600 µg / ml), until the individual clones are clearly visible.
Para el análisis por FACS se recogieron muestras a las 24, 48 y 72 horas. Posteriormente, se lavaron las células con PBS al 1% y se fijaron en paraformaldehído al 2%. Una vez fijadas las células, se analizaron y cuantificaron en el servicio de FACS del instituto del IBB-UAB.Samples were collected for FACS analysis. at 24, 48 and 72 hours. Subsequently, the cells were washed with 1% PBS and fixed in 2% paraformaldehyde. Once set the cells were analyzed and quantified in the FACS service from the IBB-UAB institute.
1 Lampe, D. J., Et al. A purified mariner transposase is sufficient to mediate transposition in vitro. Embo J 15, 5470-9. (1996).1 Lampe , DJ, Et al . A purified mariner transposase is sufficient to mediate transposition in vitro. Embo J 15, 5470-9. ( 1996 ).
2 Robertson, H. M. et al. Molecular evolution of an ancient mariner transposon, Hsmar1, in the human genome. Gene 205, 203-17. (1997).2 Robertson , HM et al . Molecular evolution of an ancient mariner transposon, Hsmar1, in the human genome. Gene 205, 203-17. ( 1997 ).
3 Oosumi, T., Belknap, W. R. & Garlick, B. Mariner transposons in humans. Nature 378, 672. (1995).3 Oosumi , T., Belknap , WR & Garlick , B. Mariner transposons in humans. Nature 378, 672. ( 1995 ).
4. Robertson, H. M. & Martos, R. Molecular evolution of the second ancient human mariner transposon, Hsmar2, illustrates patterns of neutral evolution in the human genome lineage. Gene 205, 219-28. (1997).4. Robertson , HM & Martos , R. Molecular evolution of the second ancient human mariner transposon, Hsmar2, illustrates patterns of neutral evolution in the human genome lineage. Gene 205, 219-28. ( 1997 ).
5. Reiter, L. T., Liehr, T., Rautenstrauss, B., Robertson, H. M. & Lupski, J. R. Localization of mariner DNA transposons in the human genome by PRINS. Genome Res 9, 839-43. (1999).5. Reiter , LT, Liehr , T., Rautenstrauss , B., Robertson , HM & Lupski , JR Localization of mariner DNA transposons in the human genome by PRINS. Genome Res 9, 839-43. ( 1999 ).
6. Ivics, Z., Hackett, P. B., Plasterk, R. H. & Izsvak, Z. Molecular reconstruction of Sleeping Beauty, a Tc1-like transposon from fish, and its transposition in human cells. Cell 91, 501-10. (1997)6. Ivics , Z., Hackett , PB, Plasterk , RH & Izsvak , Z. Molecular reconstruction of Sleeping Beauty, a Tc1-like transposon from fish, and its transposition in human cells. Cell 91, 501-10. ( 1997 )
7 Hartl, D. L., Lohe, A. R. & Lozovskaya, E. R. Modern thoughts on an ancyent marinere: function, evolution, regulation. Annu Rev Genet 31, 337-58 (1997).7 Hartl , DL, Lohe , AR & Lozovskaya , ER Modern thoughts on an ancyent marinere: function, evolution, regulation. Annu Rev Genet 31, 337-58 ( 1997 ).
8 Yang, Y., Greenough, K. & Wilson, J. M. Transient immune blockade prevents formation of neutralizing antibody to recombinant adenovirus and allows repeated gene transfer to mouse liver. Gene Ther 3, 412-20. (1996).8 Yang , Y., Greenough , K. & Wilson , JM Transient immune blockade prevents formation of neutralizing antibody to recombinant adenovirus and allows repeated gene transfer to mouse liver. Gene Ther 3, 412-20. ( 1996 ).
9 Zhang, L, Sankar, U, Lampe, D. J., Robertson, H. M. & Graham, F. L. The Himar1 mariner transposase cloned in a recombinant adenovirus vector is functional in mammalian cells. Nucleic Acids Res 26, 3687-93. (1998).9 Zhang , L, Sankar , U, Lampe , DJ, Robertson , HM & Graham , FL The Himar1 mariner transposase cloned in a recombinant adenovirus vector is functional in mammalian cells. Nucleic Acids Res 26, 3687-93. ( 1998 ).
10 Yant, S. R. et al. Transposition from a gutless adeno-transposon vector stabilizes transgene expression in vivo. Nat Biotechnol 20, 999-1005. (2002)10 Yant , SR et al . Transposition from a gutless adeno-transposon vector stabilizes transgene expression in vivo. Nat Biotechnol 20, 999-1005. ( 2002 )
11 Yant, S. R. et al. Somatic integration and long-term transgene expression in normal and haemophilic mice using a DNA transposon system. Nat Genet 25, 35-41. (2000).11 Yant , SR et al . Somatic integration and long-term transgene expression in normal and haemophilic mice using a DNA transposon system. Nat Genet 25, 35-41. ( 2000 ).
12 Ortiz-Urda S. et al Stable nonviral genetic correction of inherited human skin disease. Nat Med 8, 1166-70 (2002).12 Ortiz-Urda S. et al Stable nonviral genetic correction of inherited human skin disease. Nat Med 8, 1166-70 ( 2002 ).
<110> - UNIVERSITAT AUTÓNOMA DE BARCELONA<110> - UNIVERSITAT AUTONOMA DE BARCELONA
\hskip1cm- Institució Catalana de Recerca i Estudis Avançats
\ hskip1cm- Catalan Institution of Research and Study Avançats
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<120> Transposón Hsmar2 y su uso en la generación de vectores útiles en terapia génica somática.<120> Transposón Hsmar2 and its use in generation of vectors useful in somatic gene therapy.
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<130> 1302006MC<130> 1302006MC
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<210> SEQ ID NO: 1<210> SEQ ID NO: 1
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<211> 1301.<211> 1301.
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<212> ADN.<212> DNA.
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<213> Secuencia artificial<213> Artificial sequence
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<220><220>
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<223> Transposón Hsmar2.<223> Hsmar2 transposon.
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<400> 1<400> 1
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\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> SEQ ID NO: 2<210> SEQ ID NO: 2
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 42.<211> 42.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN.<212> DNA.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial.<213> Artificial sequence.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<220><220>
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido MC-D-1.<223> Oligonucleotide MC-D-1.
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 2<400> 2
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskipactgactggt cagtcaactg actggatcca ttcagctagc tg
\hfill42
\ hskip-.1em \ dddseqskipactgactggt cagtcaactg actggatcca ttcagctagc tg
\ hfill42
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> SEQ ID NO: 3<210> SEQ ID NO: 3
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 42.<211> 42.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN.<212> DNA.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial.<213> Artificial sequence.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<220><220>
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido MC-D-2.<223> Oligonucleotide MC-D-2.
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 3<400> 3
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskipcgaggggtct tcaaaaagtt catggaaaat gcgtattatg aa
\hfill42
\ hskip-.1em \ dddseqskipcgaggggtct tcaaaaagtt catggaaaat gcgtattatg aa
\ hfill42
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> SEQ ID NO: 4<210> SEQ ID NO: 4
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 42.<211> 42.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN.<212> DNA.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial.<213> Artificial sequence.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<220><220>
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido MC-D-3.<223> Oligonucleotide MC-D-3.
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 4<400> 4
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskipaaaactatgc atggatttca aaattttttg caccaaaata aa
\hfill42
\ hskip-.1em \ dddseqskipaaaactatgc atggatttca aaattttttg caccaaaata aa
\ hfill42
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> SEQ ID NO: 5<210> SEQ ID NO: 5
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 42.<211> 42.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN.<212> DNA.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial.<213> Artificial sequence.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<220><220>
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido MC-D-4.<223> Oligonucleotide MC-D-4.
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 5<400> 5
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskipctcgtactaa cttgttataa catgtctgaa caggatctag tt
\hfill42
\ hskip-.1em \ dddseqskipctcgtactaa cttgttataa catgtctgaa caggatctag tt
\ hfill42
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> SEQ ID NO: 6<210> SEQ ID NO: 6
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 42.<211> 42.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN.<212> DNA.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial.<213> Artificial sequence.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<220><220>
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido MC-D-5.<223> Oligonucleotide MC-D-5.
\newpage\ newpage
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 6<400> 6
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskiptgaggcacta agaaggataa gacatcagtt tgaaaagagc cc
\hfill42
\ hskip-.1em \ dddseqskiptgaggcacta agaaggataa gacatcagtt tgaaaagagc cc
\ hfill42
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> SEQ ID NO: 7<210> SEQ ID NO: 7
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 42.<211> 42.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN.<212> DNA.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial.<213> Artificial sequence.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<220><220>
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido MC-D-6.<223> Oligonucleotide MC-D-6.
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 7<400> 7
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskipctatcagagc aacatgaatt ctgctaaaat tgaagcaaga ac
\hfill42
\ hskip-.1em \ dddseqskipctatcagagc aacatgaatt ctgctaaaat tgaagcaaga ac
\ hfill42
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> SEQ ID NO: 8<210> SEQ ID NO: 8
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 42.<211> 42.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN.<212> DNA.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial.<213> Artificial sequence.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<220><220>
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido MC-D-7.<223> Oligonucleotide MC-D-7.
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 8<400> 8
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskipaaacatcaaa tttatggtga agcttgggtg gaagaatggt ga
\hfill42
\ hskip-.1em \ dddseqskipaaacatcaaa tttatggtga agcttgggtg gaagaatggt ga
\ hfill42
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> SEQ ID NO: 9<210> SEQ ID NO: 9
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 42.<211> 42.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN.<212> DNA.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial.<213> Artificial sequence.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<220><220>
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido MC-D-8.<223> Oligonucleotide MC-D-8.
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 9<400> 9
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskipaatcattgat gctttacgaa aagtttatgg ggacaatgcc cc
\hfill42
\ hskip-.1em \ dddseqskipaatcattgat gctttacgaa aagtttatgg ggacaatgcc cc
\ hfill42
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> SEQ ID NO: 10<210> SEQ ID NO: 10
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 42.<211> 42.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN.<212> DNA.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial.<213> Artificial sequence.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<220><220>
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido MC-D-9.<223> Oligonucleotide MC-D-9.
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 10<400> 10
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskipaaagaaatca gcagtttaca aatggataac tcgttttaag aa
\hfill42
\ hskip-.1em \ dddseqskipaaagaaatca gcagtttaca aatggataac tcgttttaag aa
\ hfill42
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> SEQ ID NO: 11<210> SEQ ID NO: 11
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 42.<211> 42.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN.<212> DNA.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial.<213> Artificial sequence.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<220><220>
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido MC-D-10.<223> Oligonucleotide MC-D-10.
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 11<400> 11
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskipgggacgagac gatgttgaag atgaagcccg cagcggcaga cc
\hfill42
\ hskip-.1em \ dddseqskipgggacgagac gatgttgaag atgaagcccg cagcggcaga cc
\ hfill42
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> SEQ ID NO: 12<210> SEQ ID NO: 12
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 42.<211> 42.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN.<212> DNA.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial.<213> Artificial sequence.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<220><220>
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido MC-D-11.<223> Oligonucleotide MC-D-11.
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 12<400> 12
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskipatccacatca atttgtgagg aaaaaattaa tcttgttcgt gc
\hfill42
\ hskip-.1em \ dddseqskipatccacatca atttgtgagg aaaaaattaa tcttgttcgt gc
\ hfill42
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> SEQ ID NO: 13<210> SEQ ID NO: 13
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 42.<211> 42.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN.<212> DNA.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial.<213> Artificial sequence.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<220><220>
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido MC-D-12.<223> Oligonucleotide MC-D-12.
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 13<400> 13
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskipcctaattgaa gaggaccgac gattaacagc agaaacaata gc
\hfill42
\ hskip-.1em \ dddseqskipcctaattgaa gaggaccgac gattaacagc agaaacaata gc
\ hfill42
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> SEQ ID NO: 14<210> SEQ ID NO: 14
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 42.<211> 42.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN.<212> DNA.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial.<213> Artificial sequence.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<220><220>
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido MC-D-13.<223> Oligonucleotide MC-D-13.
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 14<400> 14
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskipcaacaccaca gacatctcaa ttggttcagc ttacacaatt ct
\hfill42
\ hskip-.1em \ dddseqskipcaacaccaca gacatctcaa ttggttcagc ttacacaatt ct
\ hfill42
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> SEQ ID NO: 15<210> SEQ ID NO: 15
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 42.<211> 42.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN.<212> DNA.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial.<213> Artificial sequence.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<220><220>
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido MC-D-14.<223> Oligonucleotide MC-D-14.
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 15<400> 15
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskipgactgaaaaa ttaaagttga gcaaactttc cactcgatgg gt
\hfill42
\ hskip-.1em \ dddseqskipgactgaaaaa ttaaagttga gcaaactttc cactcgatgg gt
\ hfill42
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> SEQ ID NO: 16<210> SEQ ID NO: 16
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 42.<211> 42.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN.<212> DNA.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial.<213> Artificial sequence.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<220><220>
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido MC-D-15.<223> Oligonucleotide MC-D-15.
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 16<400> 16
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskipgccaaaaccg ttgcgcccag atcagctgca gacaagagca ga
\hfill42
\ hskip-.1em \ dddseqskipgccaaaaccg ttgcgcccag atcagctgca gacaagagca ga
\ hfill42
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> SEQ ID NO: 17<210> SEQ ID NO: 17
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 42.<211> 42.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN.<212> DNA.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial.<213> Artificial sequence.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<220><220>
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido MC-D-16.<223> Oligonucleotide MC-D-16.
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 17<400> 17
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskipgctttcaatg gaaattttaa acaagtggga tcaagatcct ga
\hfill42
\ hskip-.1em \ dddseqskipgctttcaatg gaaattttaa acaagtggga tcaagatcct ga
\ hfill42
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> SEQ ID NO: 18<210> SEQ ID NO: 18
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 42.<211> 42.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN.<212> DNA.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial.<213> Artificial sequence.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<220><220>
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido MC-D-17.<223> Oligonucleotide MC-D-17.
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 18<400> 18
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskipagcatttctt cgaagaattg taacaggaga tgaaacatgg ct
\hfill42
\ hskip-.1em \ dddseqskipagcatttctt cgaagaattg taacaggaga tgaaacatgg ct
\ hfill42
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> SEQ ID NO: 19<210> SEQ ID NO: 19
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 42.<211> 42.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN.<212> DNA.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial.<213> Artificial sequence.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<220><220>
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido MC-D-18.<223> Oligonucleotide MC-D-18.
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 19<400> 19
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskipttaccagtac gatcctgaag acaaagcaca atcaaagcaa tg
\hfill42
\ hskip-.1em \ dddseqskipttaccagtac gatcctgaag acaaagcaca atcaaagcaa tg
\ hfill42
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> SEQ ID NO: 20<210> SEQ ID NO: 20
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 42.<211> 42.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN.<212> DNA.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial.<213> Artificial sequence.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<220><220>
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido MC-D-19.<223> Oligonucleotide MC-D-19.
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 20<400> 20
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskipgctaccaaga ggtggaagtg gtccagtcaa agcaaaagcg ga
\hfill42
\ hskip-.1em \ dddseqskipgctaccaaga ggtggaagtg gtccagtcaa agcaaaagcg ga
\ hfill42
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> SEQ ID NO: 21<210> SEQ ID NO: 21
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 42.<211> 42.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN.<212> DNA.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial.<213> Artificial sequence.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<220><220>
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido MC-D-20.<223> Oligonucleotide MC-D-20.
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 21<400> 21
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskipctggtcaaga gcaaaggtca tggcaacagt tttttgggat gc
\hfill42
\ hskip-.1em \ dddseqskipctggtcaaga gcaaaggtca tggcaacagt tttttgggat gc
\ hfill42
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> SEQ ID NO: 22<210> SEQ ID NO: 22
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 42.<211> 42.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN.<212> DNA.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial.<213> Artificial sequence.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<220><220>
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido MC-D-21.<223> Oligonucleotide MC-D-21.
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 22<400> 22
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskiptcaaggcatt ttgcttgttg actttctgga gggccaaaga ac
\hfill42
\ hskip-.1em \ dddseqskiptcaaggcatt ttgcttgttg actttctgga gggccaaaga ac
\ hfill42
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> SEQ ID NO: 23<210> SEQ ID NO: 23
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 42.<211> 42.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN.<212> DNA.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial.<213> Artificial sequence.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<220><220>
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido MC-D-22.<223> Oligonucleotide MC-D-22.
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 23<400> 23
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskipgataacatct gcttattatg agagtgtttt gagaaagtta gc
\hfill42
\ hskip-.1em \ dddseqskipgataacatct gcttattatg agagtgtttt gagaaagtta gc
\ hfill42
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> SEQ ID NO: 24<210> SEQ ID NO: 24
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 42.<211> 42.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN.<212> DNA.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial.<213> Artificial sequence.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<220><220>
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido MC-D-23.<223> Oligonucleotide MC-D-23.
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 24<400> 24
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskipcaaagcttta gcagaaaaac gcccgggaaa gcttcaccag ag
\hfill42
\ hskip-.1em \ dddseqskipcaaagcttta gcagaaaaac gcccgggaaa gcttcaccag ag
\ hfill42
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> SEQ ID NO: 25<210> SEQ ID NO: 25
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 42.<211> 42.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN.<212> DNA.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial.<213> Artificial sequence.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<220><220>
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido MC-D-24.<223> Oligonucleotide MC-D-24.
\newpage\ newpage
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 25<400> 25
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskipagtccttctc caccacgaca atgctcctgc tcattcctct ca
\hfill42
\ hskip-.1em \ dddseqskipagtccttctc caccacgaca atgctcctgc tcattcctct ca
\ hfill42
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> SEQ ID NO: 26<210> SEQ ID NO: 26
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 42.<211> 42.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN.<212> DNA.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial.<213> Artificial sequence.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<220><220>
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido MC-D-25.<223> Oligonucleotide MC-D-25.
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 26<400> 26
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskiptcaaacaagg gcaattttgc gagagtttcg atgggaaatc at
\hfill42
\ hskip-.1em \ dddseqskiptcaaacaagg gcaattttgc gagagtttcg atgggaaatc at
\ hfill42
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> SEQ ID NO: 27<210> SEQ ID NO: 27
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 42.<211> 42.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN.<212> DNA.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial.<213> Artificial sequence.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<220><220>
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido MC-D-26.<223> Oligonucleotide MC-D-26.
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 27<400> 27
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskiptaggcatcca ccttacagtc ctgatttggc tccttctgac tt
\hfill42
\ hskip-.1em \ dddseqskiptaggcatcca ccttacagtc ctgatttggc tccttctgac tt
\ hfill42
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> SEQ ID NO: 28<210> SEQ ID NO: 28
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 42.<211> 42.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN.<212> DNA.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial.<213> Artificial sequence.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<220><220>
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido MC-D-27.<223> Oligonucleotide MC-D-27.
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 28<400> 28
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskipctttttgttt cctaatctta aaaaatcttt aaagggcacc ca
\hfill42
\ hskip-.1em \ dddseqskipctttttgttt cctaatctta aaaaatcttt aaagggcacc ca
\ hfill42
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> SEQ ID NO: 29<210> SEQ ID NO: 29
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 42.<211> 42.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN.<212> DNA.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial.<213> Artificial sequence.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<220><220>
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido MC-D-28.<223> Oligonucleotide MC-D-28.
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 29<400> 29
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskiptttttcttca gttaataatg taaaaaagac tgcattgaca tg
\hfill42
\ hskip-.1em \ dddseqskiptttttcttca gttaataatg taaaaaagac tgcattgaca tg
\ hfill42
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> SEQ ID NO: 30<210> SEQ ID NO: 30
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 42.<211> 42.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN.<212> DNA.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial.<213> Artificial sequence.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<220><220>
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido MC-D-29.<223> Oligonucleotide MC-D-29.
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 30<400> 30
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskipgttaaattcc caggaccctc agttctttag ggatggacta aa
\hfill42
\ hskip-.1em \ dddseqskipgttaaattcc caggaccctc agttctttag ggatggacta aa
\ hfill42
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> SEQ ID NO: 31<210> SEQ ID NO: 31
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 42.<211> 42.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN.<212> DNA.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial.<213> Artificial sequence.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<220><220>
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido MC-D-30.<223> Oligonucleotide MC-D-30.
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 31<400> 31
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskiptggctggtat catcgcttac aaaagtgtct tgaacttgat gg
\hfill42
\ hskip-.1em \ dddseqskiptggctggtat catcgcttac aaaagtgtct tgaacttgat gg
\ hfill42
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> SEQ ID NO: 32<210> SEQ ID NO: 32
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 42.<211> 42.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN.<212> DNA.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial.<213> Artificial sequence.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<220><220>
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido MC-D-31.<223> Oligonucleotide MC-D-31.
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 32<400> 32
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskipagcttatgtt gagaaataaa gtttatattt ttaattttta tc
\hfill42
\ hskip-.1em \ dddseqskipagcttatgtt gagaaataaa gtttatattt ttaattttta tc
\ hfill42
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> SEQ ID NO: 33<210> SEQ ID NO: 33
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 42.<211> 42.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN.<212> DNA.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial.<213> Artificial sequence.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<220><220>
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido MC-D-32.<223> Oligonucleotide MC-D-32.
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 33<400> 33
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskipttttaattcc atttttccat gaactttttg aagtcccctc g
\hfill41
\ hskip-.1em \ dddseqskipttttaattcc atttttccat gaactttttg aagtcccctc g
\ hfill41
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> SEQ ID NO: 34<210> SEQ ID NO: 34
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 42.<211> 42.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN.<212> DNA.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial.<213> Artificial sequence.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<220><220>
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido MC-R2-1.<223> Oligonucleotide MC-R2-1.
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 34<400> 34
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskipcaccctgaaa aggatccgct agccgagggg acttcaaaaa gt
\hfill42
\ hskip-.1em \ dddseqskipcaccctgaaa aggatccgct agccgagggg acttcaaaaa gt
\ hfill42
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> SEQ ID NO: 35<210> SEQ ID NO: 35
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 42.<211> 42.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN.<212> DNA.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial.<213> Artificial sequence.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<220><220>
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido MC-R2-2.<223> Oligonucleotide MC-R2-2.
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 35<400> 35
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskiptcatggaaaa atggaattaa aagataaaaa ttaaaaatat aa
\hfill42
\ hskip-.1em \ dddseqskiptcatggaaaa atggaattaa aagataaaaa ttaaaaatat aa
\ hfill42
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> SEQ ID NO: 36<210> SEQ ID NO: 36
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 42.<211> 42.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN.<212> DNA.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial.<213> Artificial sequence.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<220><220>
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido MC-R2-3.<223> Oligonucleotide MC-R2-3.
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 36<400> 36
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskipactttatttc tcaacataag ctccatcaag ttcaagacac tt
\hfill42
\ hskip-.1em \ dddseqskipactttatttc tcaacataag ctccatcaag ttcaagacac tt
\ hfill42
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> SEQ ID NO: 37<210> SEQ ID NO: 37
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 42.<211> 42.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN.<212> DNA.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial.<213> Artificial sequence.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<220><220>
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido MC-R2-4.<223> Oligonucleotide MC-R2-4.
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 37<400> 37
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskipttgtaagcga tgataccagc catttagtcc atccctaaag aa
\hfill42
\ hskip-.1em \ dddseqskipttgtaagcga tgataccagc catttagtcc atccctaaag aa
\ hfill42
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> SEQ ID NO: 38<210> SEQ ID NO: 38
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 42.<211> 42.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN.<212> DNA.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial.<213> Artificial sequence.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<220><220>
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido MC-R2-5.<223> Oligonucleotide MC-R2-5.
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 38<400> 38
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskipctgagggtcc tgggaattta accatgtcaa tgcagtcttt tt
\hfill42
\ hskip-.1em \ dddseqskipctgagggtcc tgggaattta accatgtcaa tgcagtcttt tt
\ hfill42
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> SEQ ID NO: 39<210> SEQ ID NO: 39
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 42.<211> 42.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN.<212> DNA.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial.<213> Artificial sequence.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<220><220>
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido MC-R2-6.<223> Oligonucleotide MC-R2-6.
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 39<400> 39
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskiptacattatta actgaagaaa aatgggtgcc ctttaaagat tt
\hfill42
\ hskip-.1em \ dddseqskiptacattatta actgaagaaa aatgggtgcc ctttaaagat tt
\ hfill42
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> SEQ ID NO: 40<210> SEQ ID NO: 40
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 42.<211> 42.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN.<212> DNA.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial.<213> Artificial sequence.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<220><220>
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido MC-R2-7.<223> Oligonucleotide MC-R2-7.
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 40<400> 40
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskiptttaagatta ggaaacaaaa agaagtcaga aggagccaaa tc
\hfill42
\ hskip-.1em \ dddseqskiptttaagatta ggaaacaaaa agaagtcaga aggagccaaa tc
\ hfill42
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> SEQ ID NO: 41<210> SEQ ID NO: 41
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 42.<211> 42.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN.<212> DNA.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial.<213> Artificial sequence.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<220><220>
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido MC-R2-8.<223> Oligonucleotide MC-R2-8.
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 41<400> 41
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskipaggactgtaa ggtggatgcc taatgatttc ccatcgaaac tc
\hfill42
\ hskip-.1em \ dddseqskipaggactgtaa ggtggatgcc taatgatttc ccatcgaaac tc
\ hfill42
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> SEQ ID NO: 42<210> SEQ ID NO: 42
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 42.<211> 42.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN.<212> DNA.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial.<213> Artificial sequence.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<220><220>
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido MC-R2-9.<223> Oligonucleotide MC-R2-9.
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 42<400> 42
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskiptcgcaaaatt gcccttgttt gatgagagga atgagcagga gc
\hfill42
\ hskip-.1em \ dddseqskiptcgcaaaatt gcccttgttt gatgagagga atgagcagga gc
\ hfill42
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> SEQ ID NO: 43<210> SEQ ID NO: 43
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 42.<211> 42.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN.<212> DNA.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial.<213> Artificial sequence.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<220><220>
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido MC-R2-10.<223> Oligonucleotide MC-R2-10.
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 43<400> 43
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskipattgtcgtgg tggagaagga ctctctggtg aagctttccc gg
\hfill42
\ hskip-.1em \ dddseqskipattgtcgtgg tggagaagga ctctctggtg aagctttccc gg
\ hfill42
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> SEQ ID NO: 44<210> SEQ ID NO: 44
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 42.<211> 42.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN.<212> DNA.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial.<213> Artificial sequence.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<220><220>
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido MC-R2-11.<223> Oligonucleotide MC-R2-11.
\newpage\ newpage
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 44<400> 44
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskipgcgtttttct gctaaagctt tggctaactt tctcaaaaca ct
\hfill42
\ hskip-.1em \ dddseqskipgcgtttttct gctaaagctt tggctaactt tctcaaaaca ct
\ hfill42
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> SEQ ID NO: 45<210> SEQ ID NO: 45
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 42.<211> 42.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN.<212> DNA.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial.<213> Artificial sequence.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<220><220>
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido MC-R2-12.<223> Oligonucleotide MC-R2-12.
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 45<400> 45
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskipctcataataa gcagatgtta tcgttctttg gccctccaga aa
\hfill42
\ hskip-.1em \ dddseqskipctcataataa gcagatgtta tcgttctttg gccctccaga aa
\ hfill42
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> SEQ ID NO: 46<210> SEQ ID NO: 46
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 42.<211> 42.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN.<212> DNA.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial.<213> Artificial sequence.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<220><220>
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido MC-R2-13.<223> Oligonucleotide MC-R2-13.
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 46<400> 46
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskipgtcaacaagc aaaatgcctt gagcatccca aaaaactgtt gc
\hfill42
\ hskip-.1em \ dddseqskipgtcaacaagc aaaatgcctt gagcatccca aaaaactgtt gc
\ hfill42
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> SEQ ID NO: 47<210> SEQ ID NO: 47
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 42.<211> 42.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN.<212> DNA.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial.<213> Artificial sequence.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<220><220>
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido MC-R2-14.<223> Oligonucleotide MC-R2-14.
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 47<400> 47
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskipcatgaccttt gctcttgacc agtccgcttt tgctttgact gg
\hfill42
\ hskip-.1em \ dddseqskipcatgaccttt gctcttgacc agtccgcttt tgctttgact gg
\ hfill42
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> SEQ ID NO: 48<210> SEQ ID NO: 48
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 42.<211> 42.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN.<212> DNA.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial.<213> Artificial sequence.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<220><220>
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido MC-R2-15.<223> Oligonucleotide MC-R2-15.
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 48<400> 48
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskipaccacttcca cctcttggta gccattgctt tgattgtgct tt
\hfill42
\ hskip-.1em \ dddseqskipaccacttcca cctcttggta gccattgctt tgattgtgct tt
\ hfill42
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> SEQ ID NO: 49<210> SEQ ID NO: 49
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 42.<211> 42.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN.<212> DNA.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial.<213> Artificial sequence.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<220><220>
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido MC-R2-16.<223> Oligonucleotide MC-R2-16.
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 49<400> 49
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskipgtcttcagga tcgtactggt aaagccatgt ttcatctcct gt
\hfill42
\ hskip-.1em \ dddseqskipgtcttcagga tcgtactggt aaagccatgt ttcatctcct gt
\ hfill42
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> SEQ ID NO: 50<210> SEQ ID NO: 50
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 42.<211> 42.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN.<212> DNA.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial.<213> Artificial sequence.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<220><220>
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido MC-R2-17.<223> Oligonucleotide MC-R2-17.
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 50<400> 50
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskiptacaattctt cgaagaaatg cttcaggatc ttgatcccac tt
\hfill42
\ hskip-.1em \ dddseqskiptacaattctt cgaagaaatg cttcaggatc ttgatcccac tt
\ hfill42
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> SEQ ID NO: 51<210> SEQ ID NO: 51
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 42.<211> 42.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN.<212> DNA.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial.<213> Artificial sequence.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<220><220>
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido MC-R2-18.<223> Oligonucleotide MC-R2-18.
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 51<400> 51
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskipgtttaaaatt tccattgaaa gctctgctct tgtctgcagc tg
\hfill42
\ hskip-.1em \ dddseqskipgtttaaaatt tccattgaaa gctctgctct tgtctgcagc tg
\ hfill42
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> SEQ ID NO: 52<210> SEQ ID NO: 52
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 42.<211> 42.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN.<212> DNA.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial.<213> Artificial sequence.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<220><220>
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido MC-R2-19.<223> Oligonucleotide MC-R2-19.
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 52<400> 52
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskipatctgggcgc aacggttttg gcacccatcg agtggaaagt tt
\hfill42
\ hskip-.1em \ dddseqskipatctgggcgc aacggttttg gcacccatcg agtggaaagt tt
\ hfill42
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> SEQ ID NO: 53<210> SEQ ID NO: 53
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 42.<211> 42.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN.<212> DNA.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial.<213> Artificial sequence.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<220><220>
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido MC-R2-20.<223> Oligonucleotide MC-R2-20.
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 53<400> 53
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskipgctcaacttt aatttttcag tcagaattgt gtaagctgaa cc
\hfill42
\ hskip-.1em \ dddseqskipgctcaacttt aatttttcag tcagaattgt gtaagctgaa cc
\ hfill42
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> SEQ ID NO: 54<210> SEQ ID NO: 54
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 42.<211> 42.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN.<212> DNA.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial.<213> Artificial sequence.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<220><220>
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido MC-R2-21.<223> Oligonucleotide MC-R2-21.
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 54<400> 54
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskipaattgagatg tctgtggtgt tggctattgt ttctgctgtt aa
\hfill42
\ hskip-.1em \ dddseqskipaattgagatg tctgtggtgt tggctattgt ttctgctgtt aa
\ hfill42
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> SEQ ID NO: 55<210> SEQ ID NO: 55
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 42.<211> 42.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN.<212> DNA.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial.<213> Artificial sequence.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<220><220>
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido MC-R2-22.<223> Oligonucleotide MC-R2-22.
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 55<400> 55
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskiptcgtcggtcc tcttcaatta gggcacgaac aagattaatt tt
\hfill42
\ hskip-.1em \ dddseqskiptcgtcggtcc tcttcaatta gggcacgaac aagattaatt tt
\ hfill42
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> SEQ ID NO: 56<210> SEQ ID NO: 56
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 42.<211> 42.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN.<212> DNA.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial.<213> Artificial sequence.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<220><220>
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido MC-R2-23.<223> Oligonucleotide MC-R2-23.
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 56<400> 56
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskipttcctcacaa attgatgtgg atggtctgcc gctgcgggct tc
\hfill42
\ hskip-.1em \ dddseqskipttcctcacaa attgatgtgg atggtctgcc gctgcgggct tc
\ hfill42
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> SEQ ID NO: 57<210> SEQ ID NO: 57
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 42.<211> 42.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN.<212> DNA.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial.<213> Artificial sequence.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<220><220>
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido MC-R2-24.<223> Oligonucleotide MC-R2-24.
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 57<400> 57
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskipatcttcaaca tcgtctcgtc ccttcttaaa acgagttatc ca
\hfill42
\ hskip-.1em \ dddseqskipatcttcaaca tcgtctcgtc ccttcttaaa acgagttatc ca
\ hfill42
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> SEQ ID NO: 58<210> SEQ ID NO: 58
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 42.<211> 42.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN.<212> DNA.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial.<213> Artificial sequence.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<220><220>
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido MC-R2-25.<223> Oligonucleotide MC-R2-25.
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 58<400> 58
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskiptttgtaaact gctgatttct ttggggcatt gtccccataa ac
\hfill42
\ hskip-.1em \ dddseqskiptttgtaaact gctgatttct ttggggcatt gtccccataa ac
\ hfill42
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> SEQ ID NO: 59<210> SEQ ID NO: 59
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 42.<211> 42.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN.<212> DNA.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial.<213> Artificial sequence.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<220><220>
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido MC-R2-26.<223> Oligonucleotide MC-R2-26.
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 59<400> 59
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskipttttcgtaaa gcatcaatga tttcaccatt cttccaccca ag
\hfill42
\ hskip-.1em \ dddseqskipttttcgtaaa gcatcaatga tttcaccatt cttccaccca ag
\ hfill42
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> SEQ ID NO: 60<210> SEQ ID NO: 60
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 42.<211> 42.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN.<212> DNA.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial.<213> Artificial sequence.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<220><220>
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido MC-R2-27.<223> Oligonucleotide MC-R2-27.
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 60<400> 60
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskipcttcaccata aatttgatgt ttgttcttgc ttcaatttta gc
\hfill42
\ hskip-.1em \ dddseqskipcttcaccata aatttgatgt ttgttcttgc ttcaatttta gc
\ hfill42
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> SEQ ID NO: 61<210> SEQ ID NO: 61
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 42.<211> 42.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN.<212> DNA.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial.<213> Artificial sequence.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<220><220>
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido MC-R2-28.<223> Oligonucleotide MC-R2-28.
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 61<400> 61
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskipagaattcatg ttgctctgat aggggctctt ttcaaactga tg
\hfill42
\ hskip-.1em \ dddseqskipagaattcatg ttgctctgat aggggctctt ttcaaactga tg
\ hfill42
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> SEQ ID NO: 62<210> SEQ ID NO: 62
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 42.<211> 42.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN.<212> DNA.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial.<213> Artificial sequence.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<220><220>
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido MC-R2-29.<223> Oligonucleotide MC-R2-29.
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 62<400> 62
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskiptcttatcctt cttagtgcct caaactagat cctgttcaga ca
\hfill42
\ hskip-.1em \ dddseqskiptcttatcctt cttagtgcct caaactagat cctgttcaga ca
\ hfill42
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> SEQ ID NO: 63<210> SEQ ID NO: 63
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 42.<211> 42.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN.<212> DNA.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial.<213> Artificial sequence.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<220><220>
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido MC-R2-30.<223> Oligonucleotide MC-R2-30.
\newpage\ newpage
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 63<400> 63
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskiptgttataaca agttagtacg agtttatttt ggtgcaaaaa at
\hfill42
\ hskip-.1em \ dddseqskiptgttataaca agttagtacg agtttatttt ggtgcaaaaa at
\ hfill42
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> SEQ ID NO: 64<210> SEQ ID NO: 64
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 42.<211> 42.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN.<212> DNA.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial.<213> Artificial sequence.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<220><220>
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido MC-R2-31.<223> Oligonucleotide MC-R2-31.
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 64<400> 64
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskiptttgaaatcc atgcatagtt ttttcataat acgcattttc ca
\hfill42
\ hskip-.1em \ dddseqskiptttgaaatcc atgcatagtt ttttcataat acgcattttc ca
\ hfill42
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> SEQ ID NO: 65<210> SEQ ID NO: 65
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 42.<211> 42.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN.<212> DNA.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial.<213> Artificial sequence.
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<220><220>
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido MC-R2-32.<223> Oligonucleotide MC-R2-32.
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 65<400> 65
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskiptgaacttttt gaagacccct cgcagctagc tgaatggatc ca
\hfill42
\ hskip-.1em \ dddseqskiptgaacttttt gaagacccct cgcagctagc tgaatggatc ca
\ hfill42
Claims (27)
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| ES200602688A ES2300200B1 (en) | 2006-10-18 | 2006-10-18 | TRANSPOSON HSMAR2 AND ITS USE IN THE GENERATION OF USEFUL VECTORS IN SOMATIC GENE THERAPY. |
| PCT/ES2007/000586 WO2008046943A1 (en) | 2006-10-18 | 2007-10-17 | Transposon hsmar2 and use thereof in the generation of vectors that can be used in somatic gene therapy |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| ES200602688A ES2300200B1 (en) | 2006-10-18 | 2006-10-18 | TRANSPOSON HSMAR2 AND ITS USE IN THE GENERATION OF USEFUL VECTORS IN SOMATIC GENE THERAPY. |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| ES2300200A1 true ES2300200A1 (en) | 2008-06-01 |
| ES2300200B1 ES2300200B1 (en) | 2009-05-01 |
Family
ID=39313638
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| ES200602688A Withdrawn - After Issue ES2300200B1 (en) | 2006-10-18 | 2006-10-18 | TRANSPOSON HSMAR2 AND ITS USE IN THE GENERATION OF USEFUL VECTORS IN SOMATIC GENE THERAPY. |
Country Status (2)
| Country | Link |
|---|---|
| ES (1) | ES2300200B1 (en) |
| WO (1) | WO2008046943A1 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP4136244A4 (en) * | 2020-04-13 | 2024-06-05 | Fred Hutchinson Cancer Center | Integration of large adenovirus payloads |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| ES2058738T3 (en) * | 1990-03-03 | 1994-11-01 | Degussa | TRANSPOSON, TEST VECTORS CONTAINING THIS TRANSPOSON AND PROCEDURE FOR MUTAGENESIS. |
| WO2006108525A1 (en) * | 2005-04-08 | 2006-10-19 | Max-Delbrück-Centrum für Molekulare Medizin | Reconstructed human mariner transposon capable of stable gene transfer into chromosomes in vertebrates |
-
2006
- 2006-10-18 ES ES200602688A patent/ES2300200B1/en not_active Withdrawn - After Issue
-
2007
- 2007-10-17 WO PCT/ES2007/000586 patent/WO2008046943A1/en active Application Filing
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| ES2058738T3 (en) * | 1990-03-03 | 1994-11-01 | Degussa | TRANSPOSON, TEST VECTORS CONTAINING THIS TRANSPOSON AND PROCEDURE FOR MUTAGENESIS. |
| WO2006108525A1 (en) * | 2005-04-08 | 2006-10-19 | Max-Delbrück-Centrum für Molekulare Medizin | Reconstructed human mariner transposon capable of stable gene transfer into chromosomes in vertebrates |
Non-Patent Citations (2)
| Title |
|---|
| ES 2058738 T3 DEGUSSA AKTIENGESELLSCHAFT) 11.09.1991, columna 1, líneas 58-68; columna 2, líneas 1-17,32-50; columna 3, líneas 40-48; columna 4, líneas 22-43. * |
| ROBERTSON HM & MARTOS R. Molecular evolution of the second ancient human mariner transposon, Hsmar2, illustrates patterns of neutral evolution in the human genome lineage. Gene. 1997, Vol 205 (1-2), páginas 219-228, página 221, párrafo 3.3; página 226, columnas 1-2; página 227, columna 1. * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP4136244A4 (en) * | 2020-04-13 | 2024-06-05 | Fred Hutchinson Cancer Center | Integration of large adenovirus payloads |
Also Published As
| Publication number | Publication date |
|---|---|
| WO2008046943A8 (en) | 2009-07-23 |
| ES2300200B1 (en) | 2009-05-01 |
| WO2008046943A1 (en) | 2008-04-24 |
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