WO2009106668A1

WO2009106668A1 - Vectors and uses of the mboumar transposon

Info

Publication number: WO2009106668A1
Application number: PCT/ES2009/070044
Authority: WO
Inventors: Pedro LORITE MARTÍNEZ; Teresa Amalia PALOMEQUE MESSÍA; José Antonio CARRILLO ÁVILA; Martín MUÑOZ LÓPEZ
Original assignee: Universidad De Jaén
Priority date: 2008-02-29
Filing date: 2009-02-25
Publication date: 2009-09-03
Also published as: ES2334087A1; ES2334087B1

Abstract

The invention relates to the recombinant production of a naturally active transposase of Messor bouvieri, transposon Mboumar-derived genetic constructions necessary for transgenesis and mutagenesis, and the uses thereof.

Description

VECTORS AND USES OF THE TRANSPOSON MBOUMAR

The present invention is included in the area of molecular biology, specifically within molecular genetics, and more specifically in the field of study of mobile genetic elements or transposons. It refers to the vectors necessary for the construction of genetic tools based on transposons, and their uses.

STATE OF THE PREVIOUS TECHNIQUE

Transposons are DNA sequences that can move to different positions within the genome of a cell, a process called transposition. In general they are classified into two groups:

- Class I transposons or retrotransposons, initially the retrotransposons copy themselves to RNA (transcription) but, in addition to transcribing, the RNA is copied into the DNA by a reverse transcriptase (often also encoded by the transposon) and inserted from New in the genome.

- Class II transposons, whose transposition mechanism does not imply RNA formation. They are generally transposed by a "cut and paste" process, using a transposase enzyme.

Transposases are the enzymes responsible for their mobility and are synthesized by the transposon itself. In vivo they can cause transposition of genetic material within the same chromosome, between chromosomes, and even between chromosomes and non-chromosomal genetic material, such as mitochondrial DNA. This movement of genetic material can cause serious disturbances in the organism in which it occurs, although it is also known that it has been of great importance throughout the evolution. At first the transposons were considered as "junk DNA". However, it is currently thought that they can influence the host genome in different ways. Thus, it is considered that they can modulate the gene expression by acting as promoters, activators, silencers, alternative processing sites or sites indicative of epigenetic modifications. They may also contain genes that could be included in the host genome for the performance of a specific cellular function, a process known as "molecular domestication." In addition to all of the above, due to their existence in multiple copies they can act as recombination sites originating duplications, inversions, deletions or translocations in the genome. (Charlesworth et al. 1994. The evolutionary dynamics of repetitive DNA in eukaryotes. Nature 371: 215-220; Kidwell MG. 2002. Transposable elements and the evolution of genome size in eukaryotes. Genetics 115: 49-63; Ivics & Izsvak 2004 Transposable elements for transgenesis and insertional mutagenesis in vertebrates: a contemporary review of experimental strategies Methods Mol Biol 260: 255-276.) Recent observations show that transposons can be active in somatic cells, opening the possibility that they can play an important role generating diversity between cells with the same genome. Specifically, it has been considered that this diversity could be generated by changes in the regulation of the expression of the transposase which, in turn, could affect the regulation of the gene expression of certain genes in certain cells. The effects could be beneficial (as probably occurs in cases of differentiation of neuronal cells, or in the activity of RAG proteins, important in the recombination processes that occur in the genome of lymphocytes) or deleterious (carcinogenic processes) depending on the moment of development and cell type (Collier and Largaespada. Transposable elements and the dynamic somatic genome. Rewied 2007. Genome Biology 2007, 8 (Suppl 1: S5).

Maritime transposons are sequences capable of moving around the genome that are flanked by inverted terminal repeated sequences (inverted terminal repeats or ITRs). They encode a single protein, the transposase, which, joining these ITRs, cleaves the mariner and integrates it elsewhere in the genome. The Mos1 transposase catalyzes the movement of the Mos1 transposon found in Drosophila maurítania, which is not only the first isolated mariner element, but also the first naturally active mariner element isolated. The mariner elements constitute a large family of transposons widely spread in the animal kingdom. They encode the enzyme transposase that is the only requirement for its transposition. This enzyme has two domains: the N-terminal domain or DNA binding domain and the C-terminal domain or catalytic domain, which retains the DD (34) D motif. In this enzyme, other reasons and domains essential for its performance have also been identified. Despite the aforementioned, there are still unknown aspects of its performance, whose knowledge could provide great advantages for its use as vectors, in addition to providing interesting scientific data; For this reason, in recent years numerous studies are being carried out in this regard.

The wide distribution of mariner elements, also present in humans, and their apparently easy horizontal transfer between different groups of animals, implies that they are capable of functioning in different cellular environments, that is, they do not require specific host factors to lead to Make your transposition. These facts have led to the realization of a series of studies to achieve a widely used vector in animal cells. However, most of the mariner elements that have been cloned so far are defective, since they have undergone mutations in the coding region of the transposase, so that they are unable to produce functional transposases, which has caused the transposons currently used as vectors to be mostly derived from constructions made in the laboratory, using consensus sequences. This seems to be due to a process called "vertical inactivation" (Lohe et al., 1995. Horizontal transmission, vertical inactivation, and stochastic loss of mariner-like transposable elements. Mol. Biol. Evol. 12: 62 72). Although these elements are naturally inactive (at least with respect to their transposase function), they retain their structure relatively, with several hypotheses in this regard. Thus, it may be that the inactive elements have some regulatory effect, or that they come from the recent introgression of one genome to another.

The importance of using vectors based on transposons in fields as diverse as the study of genes involved in cancer, in the generation of models of human diseases in mice, in the generation of transgenic pigs with the possibility of use as pharmaceutical farms, in Ia Gene regulation and cell differentiation, in studies of functional genomics etc., has led to the carrying out of numerous investigations with these vectors used in different organisms and in different conditions.

In vivo transposition tools have been developed in several organisms. (Table 1 shows a summary). In most cases, specific hosts required special vectors.

In vivo transposition reactions are carried out independently of the cellular or host conditions: Generally they are carried out with purified transposases, and plasmids, gene fragments, or DNA can be used as targets. genomic isolated. In successive reactions, plasmids containing transposons can be transferred to host cells.

In vitro transposition offers many advantages over in vivo systems (Devine and Boeke 1994. Efficient integration of artificial transposons into plasmid targets in vitro: a useful tool for DNA mapping, sequencing and genetic analysis. Nucleic Acids Res, 22: 3765-3772 ; Biery et al. 2000. A simple in vitro Tn7-based transposition system with low target site selectivity for genome and gene analysis. Nucleic Acids Res, 28: 1067-1077), mainly interference with host factors can be avoided. In vitro transposition reactions are generally more efficient and more flexible in the choice of DNA targets (Goryshin and Reznikoff. 1998. Tn5 in vitro transposition. J Biol Chem, 273: 7367-7374). Table 2 summarizes the different applications of transposons, both in vitro and in vivo.

The authors of this invention have described an MLE (mariner-like element) in the ant Messor bouvieri, of the order Hymenoptera, Formicidae family (Palomeque et al., Detection of a sea / ner-like element and a miniature inverted-repeat transposable element (MITE) associated with the heterochromatin from ants of the genus Messor and their possible involvement for satellite DNA evolution. 2006. Gene 371: 194-205). However, it is not known if this MLE encodes a naturally active transposase, if producing this enzyme recombinantly it would retain its possible biological activity, and if the genetic constructions carried out with its elements would be useful for in vitro transposition and / or in vivo

Since most of the transposases found are inactive, and given the large number of applications and interest that MLEs have in biotechnology, it is necessary to construct artificial transposases that are active, from the natural defects, for use as genetic tools. The most commonly used procedure consists of, from defective copies of the transposases found in various organisms in a natural way, correct the mutations that inactivate the enzyme by means of directed mutagenesis. Therefore, finding a naturally active transposase, which facilitates its production as a recombinant protein, without the need to introduce modifications in the amino acid sequence, would be a notable advance in this sector of the technique.

Table 1. Examples of transposable elements useful in transposon applications in different organisms in vivo.

Table 2. Applications of transposons.

EXPLANATION OF THE INVENTION

The authors of the invention have found that the MLE described in Messor bouvierí encodes the Mboumar transposase, which, produced in the laboratory recombinantly, is the subject of this patent application together with the vectors and other constructions necessary for its use, and it is naturally active. As indicated at different points in this application, the remaining active transposases have been reconstructed in the laboratory from defective elements present in the corresponding genomes.

The possibility of having a naturally active transposase facilitates its use and the construction of new vectors adapted to the specific needs of biotechnology and its applications.

Thus, the joint use of this transposase and the constructions made with the ITRs can constitute an in vitro transposition system that can be used in various experiments, such as the generation of random mutations. Another possible application of this system is the transgenesis and mutagenesis randomly in the genome of prokaryotic or eukaryotic cells, by insertion between the Mboumar ITRs of the desired sequence. The advantage of the use of vectors based on this type of transposable elements is their versatility, since they are not host-specific, since their only requirement is the presence of the transposase that they themselves produce.

In accordance with a first aspect of the present invention, there is provided a recombinant peptide, hereinafter peptide of the invention, consisting essentially of the amino acid sequence SEQ ID NO: 1

SEQ ID NO: 1 collects the amino acid sequence of the transposase enzyme isolated from the Messorbouvierí species.

When we compare the sequence of the Mboumar transposase (from Messor bouvieri) with the sequence of other transposases isolated in other species, it is clear that there are certain regions where they are more conserved than others, and the same occurs with the transposon. This information may be indicative that these areas are crucial to maintain the structure or function of the protein. Thus, the ITRs of different subfamilies of mariner elements (Himari, Hpmari, Hsmar2, Mos1, CsmaM, Ammari, ..), as well as Mboumar, have different regions conserved (Langin et al., 1995. The transposable element Impala, a fungal member of the Td-mariner superfamily. Mol. Gen. Genet. 246, 19-28 (Lampe et al., 2001. Loss of transposase DNA interaction may underlie the divergence of mariner family transposable elements and the ability of more than one mariner to occupy the same genome Mol. BIoI. Evol. 18, 954-961) Both the active regions determined by the specificity of the transposases and the conserved cysteines between which disulfide bridges are established, important for the conformation of the protein, will be more conserved between both proteins, while in other regions the divergence is more apparent, specifically, these enzymes have two domains: the N-terminal domain or DNA binding domain and the C-terminal domain or catalytic domain co, which retains the motive DD (34) D (Robertson HM. 1996. Members of the pogo superfamily of DNA-mediated transposons in the human genome. Mol Gen Genet 252: 761-766., Lohe et al. 1997. Mutations in the maríner transposase: the D, D (35) E consensus sequence is non-functional. Proc Nati Acad Sci USA 94: 1293-1297). Other enzymes and domains essential for its performance have also been identified in this enzyme (Pietrokovski & Henikoff 1997. A helix-turn-helix DNA-binding motif predicted for transposases of DNA transposons. Mol Gen Genet 254: 689-695., Hickman et al. 2005. Molecular architecture of a eukaryotic DNA transposase. Nat Struct Mol Biol 12: 715-721).

For all the above, it can be expected that the global identity of the transposases homologous to Mboumar, at the amino acid level, and more specifically at the level of the amino acid sequence that is collected in SEQ ID NO: 1, is 70% or more, and more preferably 80% or more and more preferably 90, or 95% or more. The correspondence between the amino acid sequence of the putative transposase (s) and the sequence of other transposases can be determined by methods known in the art. For example, those can be determined by a direct comparison of the amino acid sequence information from the putative homologous transposase, and the amino acid sequence that is collected in SEQ ID NO: 1 of this specification.

In a preferred embodiment of this aspect of the invention, the amino acid sequence of the recombinant peptide has an identity of at least 70% with SEQ ID NO: 1, preferably at least 80%, more preferably at least 90% and even more preferably at least 95%.

Peptides homologous to the Mboumar transposase of Messor bouvierí (SEQ ID NO: 1) would maintain their biological function. For example, the transposase activity includes its binding activity to the IRT and / or RDs elements ("Deleted regions"), the cleavage of the nucleic acid sequences between these IRT and / or RDs elements and its sequence insertion activity in A specific target

More preferably, these peptides homologous to the Mboumar transposase have been modified to enhance the biological activity of said enzyme. Thus, for example, amino acids can be substituted in SEQ ID NO: 1 to enhance said activity. The methods for producing such related derived sequences, for example, by site-directed mutagenesis, random mutagenesis, or enzymatic cleavage and / or nucleic acid ligation, are well known in the art, as are the methods for determine if the nucleic acid thus modified has a significant homology with the sequence that is being considered, for example, by hybridization. In another more preferred embodiment of this aspect of the invention, the recombinant peptide is homologous to the amino acid sequence of SEQ ID NO: 1.

In another aspect of the invention, the recombinant peptide of the invention is used as a transposase. In another aspect of the invention, the recombinant peptide of the invention is used as a genetic tool.

Another aspect of the invention relates to a sequence of nucleic acids or recombinant polynucleotide, hereinafter polynucleotide of the invention, which is translated to the peptide encoded by SEQ ID NO: 1, and which is selected from the list comprising: a) nucleic acid molecules that encode a peptide comprising the amino acid sequence of SEQ ID NO: 1, b) nucleic acid molecules whose complementary chain hybridizes with the polynucleotide sequence of a), or c) nucleic acid molecules whose sequence differs of a) and / or b) due to the degeneracy of the genetic code.

In a preferred embodiment of this aspect of the invention the amino acid sequence to which the polynucleotide of the invention is translated, has an identity of at least 60% with SEQ ID NO: 1. Preferably, the amino acid sequence to which the polynucleotide of the invention is translated, it has an identity of at least 80%, more preferably, of 90% and more preferably of 95%, with SEQ ID NO: 1.

In another aspect of the invention, the polynucleotide of the invention is used to produce, in amounts not yet available, the peptide of Ia invention.

Another aspect of the invention relates to a genetic construction, henceforth, the first genetic construction of the invention or aid construction, which allows the expression of the peptide of the invention, as described above, in amounts so far not available Production in quantities greater than those currently available would allow the use of the peptide of the invention as a genetic tool.

Another aspect of the invention refers to a genetic construction, hereinafter, a second genetic construction of the invention or donor construction, which comprises the ITR regions recognized by the peptide of the invention, between which a polynucleotide of interest is inserted. Additionally, it may contain a marker, such as, but not limited to, an antibiotic resistance gene.

In another aspect of the invention, a method for obtaining a recombinant peptide of the invention is described, which consists in placing the genetic construction described above in a suitable reaction medium.

This method includes the cloning and expression vectors that comprise the nucleic acid molecules of the invention. Such expression vectors include suitable control sequences, such as, for example, control elements for translation (such as start and stop codes) and for transcription (for example, promoter-operator regions, binding sites. The invention may include plasmids and viruses (comprising bacteriophages and eukaryotic viruses), in accordance with procedures well known and documented in the state of the art, and can be expressed in a variety of different expression systems, also well known and documented in the state of the art. Suitable viral vectors include baculovirus and also adenovirus and vaccine viruses. Many other viral vectors are also described in the state of the art.

A variety of techniques are known that can be used to introduce such vectors into prokaryotic or eukaryotic cells for expression, or in a germ line or in somatic cells, to form transgenic animals. Appropriate transformation or transfection techniques are well described in the literature.

The transformed or transfected eukaryotic or prokaryotic host cells, which contain a nucleic acid molecule according to the invention, as defined above, also form part of this aspect of the invention.

The expression of the sequences (same or homologous) encoding the peptide of the invention, using a series of known techniques and expression systems, including the expression in prokaryotic cells such as E.coli and in eukaryotic cells such as yeasts or the system of baculovirus-insect cell or transformed mammalian cells and in transgenic animals and plants.

In a particular embodiment of this aspect of the invention, as a vector where the Mboumar ORF is introduced, the fusion plasmid pMal-c2X (New England Biolabs) was used, together with the Escheríchia coli malE gene that encodes the binding protein to maltose (MBP: maltose-binding protein). When the tac promoter of this vector is induced in the presence of

IPTG is going to express the Mboumar + MBP transposase fusion protein that will be purified thanks to the affinity that MBP has for amylose and maltose. To control the insertion of nucleic acid molecules using the Mboumar transposon, dual transposition systems have been developed. A dual system works by contacting at least two genetic constructs (which can be, for example, two plasmids), or by means of the co-transfection or introduction of these into the same cell: the donor construct and the aid construct. The donor is a gene expression construct (promoter-gene-terminator) or a polynucleotide of interest, flanked by two ITRs (5 'ITR promoter-gene-terminator-3' ITR or 5 'ITR - polynucleotide of interest-marker - 3 'ITR or 5' ITR - polynucleotide of interest - 3 'ITR) and the aid Ia contains the Mboumar transposase under the control of a promoter. Additionally, the transposition can be carried out massively in vitro, producing the recombinant transposase exogenously, and putting it in contact, subsequently, with the donor construction.

The contact or the co-transfection of both constructions (donor and aid) in the same cell, first causes the expression of the Mboumar transposase and then the Mboumar transposase cuts the donor construction by the 2 ITRs and glue all the construction in the genome of Ia host cell, or a third recipient genetic construct, which may be, for example, a plasmid (receptor plasmid). In order to cut and paste the ITRs from the donor construction, the Mboumar-mediated transposition requires not only a complete and active Mboumar transposase in the aid construction, but also 2 binding sites for the transposase in the ITRs in the donor construction.

Another aspect of the invention relates to a method for transposition in vitro or in vivo that consists in contacting the recombinant peptide of the invention, with the genetic construction comprising the polynucleotide of interest. In a preferred embodiment of this aspect of the invention the method for transposition in vitro or in vivo is used for the generation of random mutations. In an even more preferred embodiment of the invention, the method for transposition in vitro or in vivo is used for the generation of transgenesis and random mutagenesis in the genome of prokaryotic or eukaryotic cells.

Another aspect of the invention relates to the use of the peptide of the invention for the generation of random mutations, both in vitro and in vivo. More preferably, the peptide of the invention is used for the generation of transgenesis and random mutagenesis in the genome of prokaryotic or eukaryotic cells, both in vitro and in vivo.

By insertion mutagenesis induced by Mboumar transposase, the function of certain genes could be identified, oncogenes identified and / or to introduce massive mutagenesis. To increase the number of insertional inactivations, the first ITR constructs inserted in the genome in a first generation can be massively activated by the introduction of exogenous transposases. Inactivation of genes by insertional mutagenesis with ITR would result in obtaining some mutants with the alteration of the desired phenotype. In this process, the inactivated genes are also labeled with the ITRs and their sequences can then be recovered and the genes identified.

Once the genes involved have been identified, traditional methods of genetic selection could be used to obtain lines with the desired character.

The term "recombinant peptide comprising" as used herein, refers to the fact that the peptide includes the amino acid sequence SEQ ID NO: 1, which can be integrated with other amino acids and peptides, including fusion peptides, provided it retains its function as a transposase enzyme, and which is the subject of the present invention. It also refers to amino acid sequences that encode a homologous peptide that encodes SEQ ID NO: 1.

The term "homology", as used herein, refers to the similarity between two structures due to a common evolutionary ancestry, and more specifically, to the similarity between the amino acids of two or more proteins or amino acid sequences. Since two proteins are considered homologous if they have the same evolutionary origin or if they have similar function and structure, in general, it is assumed that higher values of similarity or identity of 30% indicate homologous structures. We can consider, therefore, that identity percentages of at least 80% will maintain the transposase function of said peptide, which is the subject of the present invention.

The term "identity", as used herein, refers to the proportion of identical amino acids between two amino acid sequences that are compared. Sequence comparison methods are known in the state of the art, and include, but are not limited to, the GAG program, including GAP (Devereux et al., Nucleic Acids Research 12: 287 (1984) Genetics Computer Group University of Wisconsin, Madison, (Wl); BLASTP or BLASTN, and FASTA (Altschul et al., J. Mol. Biol. 215: 403-410 (1999). Additionally, the Smith Waterman algorithm should be used to determine the degree of identity of two sequences.

The term "fragment or derivative" of a peptide, "which possesses transposase activity" refers to fragments or peptides derived from the Mboumar transposase in their natural state that lack some or some amino acids, and that still act by transposing DNA. Alternatively, this term also refers to peptides derived from The natural Mboumar transposase, where one or more amino acids have been changed, eliminated, added, or less preferably, which has undergone investments or duplications. Such modifications are preferably made by recombinant DNA technology. Other modifications can also be made by chemical alterations of the transposase. The resulting peptide (or the fragments derived therefrom) can be produced recombinantly, and retain identical characteristics, or essentially identical to the natural Mboumar transposase.

A "recombinant peptide" as understood herein, is one that is obtained from the expression of a "recombinant polynucleotide".

The term "recombinant polynucleotide" as used herein assumes a polynucleotide of genomic, cDNA, semi-synthetic, or synthetic origin which, by virtue of its origin or manipulation: (1) is not associated with the total or a portion of a polynucleotide with which is associated in nature, (2) is linked to a polynucleotide different from that to which it is linked in nature, or (3) does not occur in nature.

The term "polynucleotide" as used herein refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. This term only refers to the primary structure of the molecule. Thus, this term includes double or single stranded DNA, as well as double or single stranded RNA. It also includes all types of known modifications (markers known in the art, methylation, auctions, replacement of one or more of the natural nucleotides with an analogue, internucleotide modifications such as, for example, those with uncharged junctions (for example, phosphonates of methyl, phosphorus tri esters, phosphorus amidates, carbamates, etc.) and with unions charged (eg, phosphorus thioates, phosphorus dithioates, etc.), those containing pendant halves, such as, for example, proteins (including, for example, nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), those with intercalating agents (for example, acridine, psoralen, etc.), those containing chelants (for example, metals, radioactive metals, boron, oxidative metals, etc.), those containing alkylating agents, those with modified bonds (for example, anomeric alpha nucleic acids, etc.), as well as unmodified forms of the polynucleotide.

The term "genetic tool" as defined herein refers to the tools used in genetic engineering for various applications, and encompasses tools of molecular genetics, quantitative genetics, population genetics, etc. In particular, in The context of the present invention refers to a tool useful for transposition in vitro, or for transgenesis and mutagenesis randomly, both in vitro and in vivo.

A "vector" is a replicon to which another polynucleotide segment has been attached, to perform the replication and / or expression of the bound segment.

A "replicon" is any genetic element that behaves as an autonomous unit of polynucleotide replication within a cell; that is, able to replicate under its own control.

"Control sequence" refers to polynucleotide sequences that are necessary to effect the expression of the coding sequences to which they are linked. The nature of such control sequences differs depending on the host organism; in prokaryotes, said control sequences generally include a promoter, a ribosomal binding site, and termination signals; in eukaryotes, generally, said control sequences include promoters, termination signals, enhancers and, sometimes, silencers. It is intended that the term "control sequences" includes, at a minimum, all components whose presence is necessary for expression, and may also include additional components whose presence is advantageous.

"Operationally linked" refers to a juxtaposition in which the components thus described have a relationship that allows them to function in the intended way. A control sequence "operatively linked" to a coding sequence is linked in such a way that the expression of the coding sequence is achieved under conditions compatible with the control sequences.

A "free reading frame" (ORF) is a region of a polynucleotide sequence that encodes a polypeptide; This region may represent a portion of a coding sequence or a complete coding sequence.

A "coding sequence" is a polynucleotide sequence that is transcribed into mRNA and / or translated into a polypeptide when under control of appropriate regulatory sequences. The limits of the coding sequence are determined by a translation start codon at the 5 'end and a translation end codon at the 3' end.

A coding sequence may include, but is not limited to mRNA, cDNA, and recombinant polynucleotide sequences.

The term "natural Mboumar", as used herein, refers to the peptide or protein encoded by the nucleotide sequence called Mboumar-9, corresponding to SEQ ID NO: 1, cloned from the genome of the Messor ant genome bouvierí. The term "Mutant Mboumar" means in this description, the protein resulting from the modification of the "natural Mboumar" protein, preferably to enhance the biological activity of said protein.

A "host" or "host cell" as used herein refers to an organism, cell or tissue that serves as a target or recipient of the transposable element to insert into themselves. A host or host cell can also indicate a cell or host that expresses a recombinant protein of interest where the host cell is transformed with an expression vector containing the gene of interest.

The term "transgenic" is used in the context of the present invention to describe animals or plants in which a non-proprietary DNA sequence introduced by a mariner element (mariner-like element MLE) has been stably incorporated into its chromosomes of such that it can pass stably to successive generations of transgenic descendants. In such circumstances, the initial transgenic organism is known as the "founder." The "founder" animal must have the non-own DNA or transgene incorporated in all its cells or in a proportion sufficient for its progeny to establish by inheritance the transgenic. When the transgene is present only in a portion of cells, the organism is referred to as a chimera. The present invention also extends to animals that incorporate the transgene stable or directly into their chromosomes and which express the transgene in their somatic cells without passing the transgene to their offspring. Specifically, these animals would serve as genetic models, for, for example, but not limited to these uses, to identify and study the function of genes, detect oncogenes, obtain biopharmaceuticals, ... Similar models described by genetic constructs derived from transposons in mice and fish zebra.

"Transgenesis" is understood herein as the process of transferring non-own DNA to an organism, which in this way becomes known as "transgenic."

Throughout the description and the claims, the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and characteristics of the invention will emerge partly from the description and partly from the practice of the invention. The following examples and drawings are provided by way of illustration, and are not intended to be limiting of the present invention.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1. Plasmid pITR and plasmid pITR-Kn. The pITR vector is obtained from the plasmid cloned with the Mboumar-9 pGEMT easy vector mariner (Promega) by the elimination of the ampicillin resistance gene (Ampr) and the origin of replication (ori). The EcoRI targets of the pGEMT easy vector have also been removed.

Figure 2. Cloning site of plasmid pITR with a cut-off region of Apo Apo I.

Figure 3. Procedure for removing EcoRI targets from pGEMT easy vector. A- Digestion with E. coli and generation of protruding ends. B- Filling the protruding ends with Klenow and generating blunt ends. C- Ligating of the two ends with T4 ligase. Figure 4. Scheme of the in vitro transposition assay.

DETAILED EXHIBITION OF REALIZATION MODES

In the ant Messor bouvierí several different copies of mariner elements have been cloned. The analysis of the nucleotide sequence of these mariner indicates that they could give rise to functional transposases, since the coding region of the transposase does not present mutations that generate stop codons, and the protein that would originate retains most of the reasons that are supposed they are necessary for the activity of the transposase.

From one of the mariner clones obtained (Mboumar-9) the transposase coding region has been isolated, which has been introduced into an expression vector to carry out its synthesis in E. coli. This transposase has been called Mboumar. Through in vitro transposition experiments it has been determined that this transposase is active, being able to mobilize the mariner element from which it comes.

Several constructions have been made through the partial elimination of the central region of the mariner, maintaining the ITRs. The deleted region has been replaced by a marker gene, which in this case is the kanamycin resistance gene (Kn). Through in vitro transposition it has been proven that this construction (ITR-Kn -ITR gene) can be mobilized by the Mboumar transposase, and, therefore, any other sequence placed between these ITRs.

Next, the invention will be illustrated by tests carried out by the inventors, which shows the activity of the recombinant peptide of the invention and the effectiveness of the genetic constructs and of said peptide in the in vitro transgenesis. a) Purification of the Mboumar transposase

The in vitro production of the protein is carried out by introducing its coding region (ORF) into an expression vector. Of the multiple expression vectors available on the market, the fusion plasmid pMal-c2X (New England Biolabs) has been used in this case.

The Mboumar ORF is introduced into the pMal-c2X vector downstream of the Escherichia coli malE gene encoding the maltose binding protein (MBP: maltose-binding protein). When the tac promoter of this vector is induced in the presence of IPTG, the Mboumar + MBP transposase fusion protein will be expressed, which will be purified thanks to the affinity that MBP has for the amylose and the maltose.

The Mboumar transposon ORF is amplified from the Mboumar-9 clone (Palomeque et al. 2006. Detection of a mariner-Wke element and a miniature inverted-repeat transposable element (MITE) associated with the heterochromatin from ants of the genus Messor and their possible involvement for satellite DNA evolution (Gene 371: 194-205) with two primers that incorporate two cutting targets for the restriction enzymes EcoRI and BamHI, which will allow it to be introduced in phase in the polylinker of pMal-c2X. Once the plasmid that includes the Mboumar ORF at the correct cloning site in pMal-c2X is obtained, it is introduced by transformation into E. coli bacteria of the Rosetta 2 strain. With one of the positive colonies obtained 100 ml of inoculated liquid culture of LB medium with ampicillin (Amp), and incubated under stirring at 37 ⁰ C until a a = 0.5 is obtained. IPTG is then added to a final concentration of 0.1 mM and kept under stirring overnight at room temperature. The culture is then centrifuged at 5,000 rpm for 10 minutes. The precipitate is resuspended in 10 ml of HSG buffer (Hepes pH = 7.5 50 mM, 200 mM NaCI, 5 mM EDTA, 2 mM DTT and 10% glycerol) to which protease inhibitor is added. The 100 ml of culture is introduced in a French press and the bacteria are subjected to a pressure of 1,000 Psi. The lysate obtained is centrifuged at 15,000 rpm for 30 minutes. The supernatant is passed through a column with 1 ml of amylase (previously washed with Buffer HSG) where the fusion protein will be retained. Once all the supernatant has passed through the amylose matrix, it is washed again with Buffer HSG. Finally, to separate the protein, 1 ml of Buffer HSG with maltose is added at a concentration of 10 mM, diluted in buffer A (Hepes pH = 7.5 50 mM, 50 mM NaCI, 5 mM EDTA, 2 mM DTT and a 5% glycerol) and is passed through an ion exchange column (FPLC) taking several fractions. These fractions are migrated on an SDS-Page gel and the one with a higher proportion of complete protein is selected with respect to the incomplete one. To the selected fraction glycerol is added (final concentration of 10%), aliquot and stored at -8O ⁰ C for later use. The protein concentration is quantified by the Bradford method.

The sequence of the Mboumar-9 transposase, obtained from the DNA sequence of the Mboumar-9 mariner, is collected in SEQ ID NO: 1.

b) Basis of in vitro transposition

To determine the in vitro activity of the transposase it is necessary to develop two vectors, one the donor and the other the recipient. The Donor Plasmid will contain the sequence to be mobilized, which must be flanked by the ITRs of the Mboumar transposon. The Receptor Plasmid can be any other where the insertion of the sequence located between the two ITRs provided by the donor plasmid can be detected. During the in vitro transposition the Mboumar transposase will recognize the ITRs that are located in the donor plasmid. The transposase will make a cut at the ends of these ITRs and integrate this DNA fragment into a TA dinucleotide (which will undergo duplication during the process) of the recipient plasmid. However, it is also possible that integration takes place at a site other than the donor plasmid itself. To avoid this inconvenience, the donor plasmid must be unable to replicate in the bacteria that will be transformed with the reaction of the transposition in vitro.

c) Design of vectors for in vitro transposition

Two donor plasmids have been constructed: pITR, which presents the ends of the mariner element and therefore its ITRs, and the pITR-Kn, derived from the previous one, but to which the kanamycin resistance gene (Knr gene) has been incorporated ( Fig. 1).

c.1. PITR plasmid

For the construction of this vector, the Mboumar-9 plasmid containing the Mboumar-9 mariner element cloned in the plasmid pGEMT easy vector (Promega) is used. The first modification that is made on the plasmid is the elimination of the ampicillin resistance gene (Ampr) and the origin of replication (ori). For this, the plasmid has been digested with the Seal and Accl enzymes, thus releasing most of the Ampr gene and the ori. This fragment is replaced by the origin of R6K replication, which will allow this plasmid to replicate only in pir + bacteria, but not in pir- strains, which will be those transformed with the transposition reaction. Subsequently, the plasmid is digested with the restriction enzymes Mscl and Ndel (partial digestion), and the ends generated in the vector are joined by ligation. These enzymes will release the central region of the mariner leaving only 173 bp of the ends, in which the two ITRs are found. In this region a target sequence for the Apol enzyme is located, which can be used to insert a DNA sequence (Fig. 2). However, this enzyme is also able to cut on the EcoRI targets found in the pGEM-T vector, on both sides of the insert, so it is necessary to eliminate them.

To eliminate the EcoRI targets, the plasmid is digested with this enzyme, generating two fragments, one with the insert corresponding to the mariner and the other with the vector. This enzyme generates protruding ends that are filled with the help of the Klenow enzyme, thus generating fragments with blunt ends that are re-bound, thus degenerating the target for EcoRI (Fig. 3), leaving the Apol target between the two ITRs as a single target for this enzyme in the plasmid (Fig. 2)

c.2. PITR-Kn plasmid

Plasmid pITR-Kn is derived from the pITR to which the kanamycin resistance gene (Knr gene) has been inserted into the Apol target (Fig. 1)

c.3. Receptor plasmid

As a receptor plasmid, any other that confers resistance to an antibiotic other than kanamycin and is capable of replicating in pyr- bacteria can be used. For the in vitro transposition assays the pGEMT Easy Vector (Promega) vector itself has been used.

d) In vitro transposition test (Figure 4) • Elements necessary for in vitro transposition:

Donor plasmid (pITR-Kn).

Plasmid receptor (pGEM-T). - Purified Mboumar transposase.

Reaction buffer: It is the buffer in which the necessary conditions are met for the transposition by the Mboumar transposase to be carried out.

• In vitro transposition reaction:

Donor plasmid: 9 μM.

Receiving plasmid: 9 μM.

Mboumar transposase: 10 nM. - 1x reaction buffer:

Glycerol: 10%.

Hepes (pH = 7.9): 25 mM.

BSA: 12.5 μg / μl.

DTT: 2 mM. - NaCMOO mM.

MgCl2: 10 mM.

This reaction is incubated at 28 ⁰ C place overnight. During this period the transposase cleaves the Knr gene along with the ITRs that flank it and integrate it into TA dinucleotide of the recipient plasmid (or donor plasmid). The mobilization of this sequence to the recipient plasmid will generate a plasmid that will confer resistance to kanamycin and ampicillin, and that will be able to replicate in pyr- bacteria.

Then, competent bacteria pir- (DH5α) are transformed with 8 µl of the transposition reaction and the negative control (without transposase). The half of the volume of the transformation with the transposition reaction is seeded in LB plates with kanamycin and ampicillin, and the other half in plates with only ampicillin. The same volume is the one that is going to be sown of the transformation with the negative control in an LB plate + ampicillin + kanamycin.

In the plates with only ampicillin, all those bacteria that have incorporated the original receptor plasmid or with the insertion of the Kn ^r gene can grow, since the receptor plasmid contains the Amp ^r gene.

In the plates with ampicillin and kanamycin only those bacteria that have the receptor plasmid with the insertion of the Kn ^r gene can grow, as long as the integration has occurred outside the Amp ^r gene and the origin of replication {orí). The integration in any of these sites would give rise to non-viable bacteria, since the integration in the Amp ^r gene would make the bacteria not resistant to ampicillin, and the integration in the ori would prevent the replication of the receptor plasmid within the bacterium. .

Bacteria that have incorporated the donor plasmid (with or without the Kn ^R gene) will not survive, since this plasmid is not able to replicate in the pir-DH5α bacteria.

• Calculation of Transposition Efficiency:

The efficiency with which the Mboumar transposase has carried out the transposition under the conditions described is 10 ^{~ 3} . This is calculated by dividing the number of Transformer Receptor Plasmids with the Kn ^R gene integrated by the total number of Transformer Receptor Plasmids.

Claims

1. Recombinant peptide comprising the amino acid sequence SEQ ID NO: 1

2. Recombinant peptide according to claim 1 whose amino acid sequence has at least 70% identity with SEQ ID NO: 1

3. Recombinant peptide according to claim 1 that has an identity of at least 80% with the amino acid sequence of SEQ ID NO: 1.

4. Recombinant peptide according to claim 1 that has an identity of at least 90% with the amino acid sequence of Ia

SEQ ID NO: 1.

5. Recombinant peptide according to claim 1 that has an identity of at least 95% with the amino acid sequence of SEQ ID NO:!

6. Peptide homologous recombinant peptide according to any of claims 1-5.

7. Isolated polynucleotide, which translates to the amino acid sequence of SEQ ID NO: 1, and is selected from the list comprising: a. nucleic acid molecules that encode a polypeptide comprising the amino acid sequence of SEQ ID NO: 1, b. nucleic acid molecules whose complementary hybrid chain with the polynucleotide sequence of a), or C. Nucleic acid molecules whose sequence differs from a) and / or b) due to the degeneracy of the genetic code. for use in the production of the recombinant peptide according to any of claims 1-6.

8. Polynucleotide according to claim 7, wherein the amino acid sequence to which it is translated has an identity of at least 60% with SEQ ID NO: 1.

9. Polynucleotide according to claim 7, wherein the amino acid sequence to which it is translated has an identity of at least 80% with SEQ ID NO: 1.

10. Polynucleotide according to claim 7, wherein the amino acid sequence to which it is translated has an identity of at least 90% with SEQ ID NO: 1.

11. Polynucleotide according to claim 7, wherein the amino acid sequence to which it is translated has an identity of at least 95% with SEQ ID NO: 1.

12. Polynucleotide according to any of claims 7-11, which encodes a peptide according to any of claims 1-6.

13. Genetic construction comprising: a. a vector, b. the polynucleotide according to any of claims 7-12, and c. a control sequence operatively linked, in such a way that it allows the expression of the recombinant peptide according to any of claims 1-6.

14. Genetic construction comprising: a. a vector, b. the ITR regions recognized by the recombinant peptide according to any of claims 1-6, and c. a polynucleotide inserted between the ITR regions of step b)

15. Method for obtaining a recombinant peptide according to any of claims 1-6 comprising: a. Entering a genetic construct according to claim 13 in a host cell. b. Incubate the host cell according to a. in a suitable reaction medium. C. Purify the recombinant peptide obtained.

16. Method for in vitro transposition comprising contacting the recombinant peptide according to any of claims 1-6, or the peptide obtained according to the method described in claim 15, with the genetic construction according to claim 14.

17. Method according to claim 16, for the generation of random mutations.

18. Method according to claim 16 for the generation of transgenesis and random mutagenesis in the genome of prokaryotic or eukaryotic cells.

19. Use of the recombinant peptide according to any of claims 1-6, or a fragment or derivative thereof, as a transposase.

20. Use of the recombinant peptide according to any of claims 1-6, as a genetic tool.

21. Use of the recombinant peptide, according to any of claims 1-6, for the generation of random mutations in vitro.

22. Use of the recombinant peptide, according to any of claims 1-6, for the generation of transgenesis and random mutagenesis in vitro, in the genome of prokaryotic or eukaryotic cells.