ES2338971B1 - ANIMAL MODEL FOR THE STUDY OF LIVING ANGIOGENESIS AND LYMPHANGIOGENESIS. - Google Patents

Abstract

Bad animal model for the study of angiogenesis and lymphangiogenesis in vivo .

The present invention is within the field of molecular biology and biotechnology, and refers to a genetically modified non-human mammal whose cells integrate IRES-EGFP-Luciferase construction in the 3'UTR region of the Flt4 gene, and that is able to report the expression of the Flt4 gene with an identical pattern to the expression of the VEGFR-3 receiver. Said non-human mammal is useful in the study and evaluation of the progression of diseases that They have lymphangiogenesis, angiogenesis and / or inflammation, as well as for the trial of drugs aimed at modulating these processes, and by Therefore, for the development of antiangiogenic therapies, antitumor and antimetastatic.

Description

Animal model for the study of angiogenesis and lymphangiogenesis in vivo .

The present invention is within the field of molecular biology and biotechnology, and refers to a genetically modified non-human mammal whose cells integrate IRES-EGFP-Luciferase construction in the 3'UTR region of the Flt4 gene, and that is able to report the expression of the Flt4 gene with an identical pattern to the expression of the VEGFR-3 receiver. Said non-human mammal is useful in the study and evaluation of the progression of diseases that They have lymphangiogenesis, angiogenesis and / or inflammation, as well as for the trial of drugs aimed at modulating these processes, and by Therefore, for the development of antiangiogenic therapies, antitumor and antimetastatic.

Prior art

The Flt4 gene encodes for the receptor VEGFR-3, a membrane receptor with activity tyrosine kinase that belongs to the family of vascular endothelial growth factor receptors (VEGF A-D). This family of receptors (VEGFR 1-3) has a relatively expression pattern specific endothelial cells of blood and lymph vessels and they are very important regulators of the angiogenesis processes and neovascularization, as they participate in the control of proliferation, migration and differentiation of cells endothelial

The VEGFR-3 receiver begins to express yourself in the mouse during embryonic development approximately on day 8 of gestation in the blood vessels primordial, but its expression is restricted to lymphatic vessels at the moment they develop. In fact, on day 12 of embryonic development the expression of VEGFR-3 is already mainly detected in the lymphatic vessels (our own results). In the adult animal, it has been described that in conditions physiological VEGFR-3 is expressed mostly in endothelial cells of lymphatic vessels, and is considered a good marker of this cell type because unlike the receptors VEGFR-1 and -2, VEGFR-3 is not found expressed in the endothelium of adult blood vessels, under physiological conditions

Although specific for endothelial cells lymphatic, the expression of VEGFR-3 in adults is low in baseline conditions. In the mouse a fall is observed dramatic expression levels among young animals (1-5 weeks) and adult animals (more than 6 weeks)

In general, there is a correlation both at the level spatial as temporal between the expression of VEGF receptors and angiogenesis processes. Thus, in adults the expression of these receptors is increased associated with scarring tissue, inflammation, tumor growth and metastasis formation, all of them, processes that depend on the formation and growth of New glasses from existing ones.

There is currently a model developed by Xenogen Corporation based on VEGFR-2 receptor expression signals (Flk1 / KDR) (Zhang et al ., 2004. Blood , 103, 617-626. US 6,867,348). It is a genetically modified mouse in which two fragments of the mouse Flk1 gene containing the promoter and enhancer sequences respectively have been cloned into a plasmid containing the sequence encoding the luciferase enzyme. This construct has been introduced into the mouse genome at random by microinjection into pronuclei of embryos of a cell.

In addition, other reporter models have been developed based on the expression signals of other genes whose expression is specific to endothelial cells. Two transgenic lines have been developed by different laboratories in which the EGFP ( enhanced green fluorescent protein ) gene is expressed under the control of the promoter or promoter / enhancer of the Tie1 receptors (Iljin et al ., 2002. Faseb J , 16 , 1764-1774) and Tie2 (Motoike et al ., 2000; Genesis , 28, 75-81) respectively. Tie1 and Tie2 are expressed during embryonic development in the mouse, in angioblasts and in endothelial cells. In the adult organism the endogenous expression of these receptors in the endothelium is maintained at lower levels, and is induced in neovascularization sites such as the ovary during the formation of the corpus luteum and in healing processes. However, the expression of reporter genes under the control of regulatory sequences of these genes in the corresponding genetically modified models, such as the Tie1-EGFP line is not induced in the endothelium during tumor neovascularization. This induction has been observed, however, by replacing part of the endogenous Tie1 gene sequence with the lacZ gene, as was done for the Tie1 knockout generation (Puri et al ., 1995). The discrepancy between the behavior of the knockout / knockin line and the transgenic Tie1 line regarding the activation of Tie1 expression during tumor angiogenesis reflects the limitation of conventional transgenic lines to faithfully reproduce the regulation of gene expression endogenous The same is observed in a transgenic line expressing lacZ under the control of the VEGFR-3 promoter, in which the endogenous expression of the receptor is not fully reproduced (lljin et al ., 2001).

Description of the invention

The inventors have constructed an animal model, a genetically modified mouse by means of gene-targeting ( knock-in ) in which a tracer or reporter protein will be expressed under the control of the VEGF-3 receptor expression signals. The expression of this reporter can be monitored in vivo by non-invasive techniques, which implies that angiogenesis associated with different processes, both physiological and pathological in real time, could be measured and quantified directly in the mouse.

Therefore, a first aspect of the invention is refers to a genetically modified non-human mammal, from now on hereinafter mammal of the invention, whose cells express the IRES-EGFP-Luciferase construction in the 3'UTR region of the Flt4 gene with an identical pattern to the expression of the VEGFR-3 receiver, both in space and in the weather.

In a preferred embodiment of this aspect of the invention the expression of the construction IRES-EGFP-Luciferase comprises the introduction of a nucleic acid sequence comprising the SEQ ID NO: 1 in the animal or one of its ancestors.

The sequence SEQ ID NO: 1 comprises the IRES sequences of the encephalomyocarditis virus, the sequence of EGFP and luciferase.

The IRES sequences (from the English " internal ribosome entry site ") are nucleotide sequences that allow the initiation of protein synthesis in the medium of translating the open reading frame of a messenger RNA (mRNA or mRNA). Unlike the best known mechanism of protein translation in eukaryotic organisms that requires a previous modification at the 5 'end of the messenger mRNA for the assembly of the translation machinery, the IRES sequences are recognized by the 43S pre-initiation complex, so that can start the translation of messenger RNA despite lacking Cap modification at its 5 'end. These sequences have been found in members of the viral families picornavirus, retrovirus and herpesvirus. In addition, IRES sequences have recently been found in mRNA of eukaryotic organisms, especially in proteins involved in cell cycle regulation, as well as apoptotic mechanisms. In the context of the present invention, IRES sequences are used, but not limited to, the IRES sequence of the encephalomyocarditis virus.

The EGFP according to its acronym in English ( enhanced green fluorescent protein ) is a mutation of a protein (GFP) produced by the jellyfish Aequorea sp . ( A. victoria, A. aequorea, A. forskalea ) that emits bioluminescence in the green zone of the visible spectrum. This mutation (S65T) increases fluorescence, photostability and has excitation and emission peaks compatible with fluorescein isothiocyanate filters (FITC), so the gene encoding this protein is isolated and is commonly used in molecular biology as marker.

In this report "luciferase" refers to a generic term that groups a group of enzymes capable of producing bioluminescence in nature. The best known is the firefly luciferase , from Photinus pyralis . In luminescence reactions, light is produced by the oxidation of luciferin (a pigment), and the reaction requires ATP (adenosine triphosphate). Here, therefore, "luciferase" and "luciferin" do not refer to specific molecules, but rather generic terms for a substrate and its associated enzyme.

In this report it is defined as the "Flt4" gene, "FMS-like tyrosine kinase 4", "VEGFR3", "VEGFR-3", "Chy" and / or "AI323512" a nucleotide or polynucleotide sequence, which constitutes the coding sequence of the Flt4 polypeptide, and which would comprise various variants from:

to)

nucleic acid molecules that encode a polypeptide comprising the amino acid sequence of SEQ ID NO: 2,

b)

nucleic acid molecules whose chain complementary hybrid with the polynucleotide sequence of to),

C)

nucleic acid molecules whose sequence differs from a) and / or b) due to code degeneration genetic,

d)

nucleic acid molecules that encode a polypeptide comprising the amino acid sequence with an identity of at least 80%, 90%, 95%, 98% or a 99% with SEQ ID NO: 2.

wherein the polypeptide encoded by said nucleic acids have the activity and characteristics Structural Flt4 protein.

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The genetic modification of the Flt4 gene that performed in the invention leads to the expression of a messenger bicistronic, which is expressed using the regulation signals Endogenous of the Flt4 gene. The translation of this messenger results simultaneously to the synthesis of two proteins: the receptor VEGFR-3 intact and fusion protein EGFP-luciferase. Therefore, after the genetic modification, the EGFPluc reporter expresses recapitulating exactly the same endogenous expression pattern of the Flt4 gene, with identical control both spatially and temporally.

The use of fusion EGFP-luciferase as a reporter molecule, allows monitor VEGFR3 expression using imaging techniques optics both by fluorescence emitted by the EGFP and by luminescence emitted by luciferase in the presence of its sutrate specific, luciferin.

The photonic emission that is emitted when the luciferase reacts with the appropriate luciferin can be detected by a light-sensitive device called a luminometer, or by modified optical microscopes, or allowing the observation of biological processes, even in vivo , in the animal Complete previously anesthetized.

As used herein, the term "non-human mammal" refers to a non-human mammalian animal of any genetic background, preferably laboratory animals such as rodents, more preferably rats and mice, or non-human primates. Said non-human animal can have practically any known genetic background, that is, it can be a wild-type or wt animal or it can be combined with other genetic alterations in the same mouse, for example, a transgenic animal , mutant or deficient ( knock-out or KO), with spontaneous or experimentally induced mutations, likewise in any genetic background.

The term "transgenic", applied to non-human mammal, as used in the present invention, is refers to mammals that contain a transgene and includes, by way of illustrative and without limiting the scope of the present invention, to conventional transgenic animals, or obtained by recombination Homologue: KOs, KIs, conditional models etc.) in any fund genetic.

The authors of the present invention have introduced the Flt4 knock-in modified allele, Flt4-IRES-EGFPLuc, into an immunosuppressed mouse (nu / nu). This genetic background provides the advantage of allowing xenografts to be tested with tumor lines of different origins, both murine and human, and to quantify angiogenesis / lymphangiogenesis in relation to the size of tumors by combining different imaging techniques in vivo . Likewise, using metastatic lines, the angiogenesis associated with the establishment of metastases in this model could be quantified.

The added advantage of using a nude background (nu / nu) is that in nude mice masking does not occur of the signal emitted by luciferase as a result of the interference of the animal's hair, with which the signal is more Intense and allows better detection and quantification of changes weak expression. Therefore, in an even more realization preferred of this aspect of the invention, the non-human mammal is an immunosuppressed mouse (nu / nu).

The reporter molecule (the fusion EGFP-luciferase) is expressed under the control of endogenous expression signals of the Flt4 gene and therefore has a expression pattern identical to that of the recipient both in space As in time. This does not happen in transgenic models conventional.

The insertion of the cassette reporter is done in the 3 'untranslated region of the gene (3'UTR, from the unstranlated English region or from an untranslated trailer ), outside the coding sequence, so the coding sequence of the Flt4 gene is not found altered In models in which the gene coding sequence is replaced by a marker, the marker does recapitulate the endogenous expression pattern of the corresponding gene but simultaneously in these models a copy of the gene is inactivated which in many cases can lead to associated phenotype that can manifest even in heterozygosis with the second copy of the gene intact (haploinsufficiency).

Here, a " cassette " or "cassette" is a coding region of a gene from a prokaryotic or eukaryotic organism flanked by the regulatory elements necessary for its expression in vivo or in vitro . Although expression cassettes can have very varied configurations, they must contain at least one promoter ( promoter ), a coding region (eukaryotic cDNA or prokaryotic gene) and a transcription terminator (terminator) or a polyadenylation site, as appropriate of a gene derived from a prokaryotic organism or a cDNA from a eukaryotic organism. To this basic configuration is added, if necessary, a sequence with regulatory function for the natural expression of the gene in the chosen system, eg: an operator, an enhancer, the Shine and Dalgarno sequence for binding to the rRNA of E. coli , or the sequences of a signal peptide (if the protein is exported).

In another aspect of the invention, the mammal of the invention has the construction introduced IRES-EGFP-Luciferase 149 nucleotides below the TGA stop codon within the 3'UTR region of the gene Flt4.

The reporter gene, when introduced into the region 3'UTR of the Flt4 gene, between the stop codon and the sequence of polyadenylation, does not affect the normal expression of this gene, so that the animal model can be used in both heterozygosis and in homozygosis. This provides the advantage of being able to control the signal baseline fluorescence / luminescence according to the requirements of the trial, without this having any consequences on the phenotype of the Model to study.

Humans and laboratory mice (both euterian mammals) have more genes in common than could be found among humans and non-euterian animals. The genetic resemblance between the two species makes it possible to compare genes almost directly, and allows scientists to find the same genes in humans to decode the routes and mechanisms of human diseases, because mice can also develop them. Therefore, in another preferred embodiment, the mammal of the invention is a mouse ( Mus musculus ).

Advantageously, gene construction IRES-EGFP-Luciferase is included within a vector, such as, for example, an expression vector or A transfer vector. As used herein invention the term "vector" refers to systems used in the process of transferring an exogenous gene or a exogenous gene construction inside a cell, allowing in this way the stable vehicle of genes and constructions exogenous gene Such vectors may be non-viral vectors or viral vectors.

Thus, in another aspect of the invention there is provided a gene-targeting vector, hereafter referred to as the vector of the invention, comprising the construction containing the cDNA encoding the EGFP-luciferase fusion protein, preceded by a sequence of internal ribosome entry (IRES), the genomic sequences of the Flt4 locus that flank the integration point of the IRES-EGFP-luciferase construct in the 3'UTR region of the Flt4 gene, and a positive selection cassette comprising a phosphoglycerate promoter kinase (PGK in Fig. 1), a neomycin resistance gene (NEO in Fig. 1) and a polyA signal from the SV40 virus, flanked by frt sequences.

In a preferred embodiment, the vector of the invention is further accompanied by a negative selection cassette comprising the mouse phosphoglycerate kinase gene promoter, the coding sequence of the herpes simplex virus thymidine kinase gene (TK in the Fig. 1), and a polyA signal of the SV40 virus.

Another aspect of the invention relates to a procedure for obtaining the mammal of the invention, in hereinafter method of the invention, comprising:

to)

build a vector of the invention as described above,

b)

transfect said construction in a embryonic stem cell population, and select cells embryonic mothers who have stably integrated the vector of the invention in its genome by homologous recombination.

C)

incorporate the stem cell clones embryonic recombinants from step b) to host embryos, so that recombined cells contribute to all lineages embryo cell,

d)

allow embryo development to obtain a chimeric non-human mammal with the allele that expresses the IRES-EGFP-Luciferase construction in the 3'UTR region of the Flt4 gene.

and)

cross the chimeric non-human mammal from step d) to produce heterozygous mammals for construction expression IRES-EGFP-Luciferase in the region 3'UTR of the Flt4 gene,

F)

cross heterozygous individuals according to section e) with transgenic animals that express the Flp recombinase ubiquitously in all cells including the germ line (Tg-pCAG Flp) to eliminate the pGK-neo cassette flanked by sites Frt,

and optionally,

g)

cross heterozygous individuals according to section f) each other to produce a non-human mammal genetically modified homozygous for the expression of the IRES-EGFP-Luciferase construction in the 3'UTR region of the Flt4 gene.

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In a preferred embodiment of the method of invention the embryonic stem cells are genetic background V6.4 F1 hybrid (C57BL / 6 x 129).

As used in the present invention the term "transfect" refers to any technique or procedure that allows integration into a series of cells a living organism of an exogenous gene, or "transgene", and that confers on these cells and the organism that carries them a new biological property Preferably the transfection method is the electroporation Said transgene or exogenous gene refers to a DNA normally not resident, nor present in the cell that is intended transform. In the particular case of the present invention the DNA it codes for EGFP and for a luciferase. On the other hand, the transgenesis process to obtain the animal model of the invention can be applied to both animals completely developed as to embryos thereof as long as it allows construction integration IRES-EGFP-Luciferase in the region 3'UTR of the Flt4 gene. Preferably, the transgenesis process is Applied to embryos.

Another aspect of the invention relates to a cell, hereinafter cell of the invention, isolated from mammal of the invention or that has integrated the vector of the invention by homologous recombination. A preferred embodiment of this aspect of the invention relates to the derived cell line of the cell or cells of the invention.

The mammal of the invention is useful in the non-invasive monitoring of angiogenesis in tumors and other pathologies and their application to the development of therapies antiangiogenic It also has a universal utility for study of any process associated with operation and development of the endothelium, such as inflammatory processes, vascular pathologies and particularly, tumor growth, cell extravasation Tumor and metastasis establishment.

The use of the model as a reporter of lymphangiogenesis / angiogenesis / inflammation also implies its use for the trial of drugs aimed at modulating these processes and therefore for the development of therapies antiangiogenic, antitumor and antimetastatic.

Thus, another aspect of the invention is refers to a method of detecting potentially useful agents in the treatment or prevention of inflammatory processes that understands:

to)

induce an inflammatory and / or angiogenic process in the mammal of the invention,

b)

recombinantly express the agent, or administering the agent to the mammal of the invention of a),

b)

compare the emission level of and / or luminescence in the mammal of the invention of a) with:

i.

the level of fluorescence emission and / or luminescence in the same mammal of the invention before the compound administration,

ii.

the level of fluorescence emission and / or luminescence in another mammal of the invention, which has not been administered the compound; or

iii.

a standard level of fluorescence emission and / or luminescence for mammals according to i or ii;

where a favorable change with the level of indicator (decrease in fluorescence and / or luminescence) in relationship with the reference change using normal animals not afflicted by inflammatory processes under comparable conditions, indicates that the compound is active to treat said process inflammatory.

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Induction of the inflammatory process and / or Angiogenic in the mammal of the invention can be performed by various media, including, but not limited to, inflict a wound, administer an angiogenic agent, induce a tumor process, xenografts in a nude model (nu / nu).

In the present invention, it is understood by "agent" any chemical compound of natural origin or synthetic, with a genetic therapeutic activity or an activity Pharmacological Therapeutics The concepts of gene therapy as well as The pharmacological therapy are well known to an expert in the matter.

In a preferred embodiment of this aspect, the mammal of the invention is a mouse. In an even more preferred embodiment is a nude mouse (nu / nu). In an even more preferred embodiment, the introduction of the IRES-EGFP-Luciferase cassette is performed 149 nucleotides below the TGA stop codon within the 3'UTR region of the Flt4 gene. And in another even more preferred embodiment, the mammal of the invention is homozygous for the introduction of the IRES-EGFP-Luciferase cassette into the 3'UTR region of the Flt4 gene.

Another aspect of the invention relates to a method of detecting potentially useful agents in the Treatment or prevention of tumor processes comprising:

to)

induce a tumor process in the mammal of the invention,

b)

recombinantly express the agent, or administering the agent to the mammal of the invention,

b)

compare the level of fluorescence emission and / or luminescence in the mammal of the invention of a) with:

i.

the level of fluorescence emission and / or luminescence in the same mammal of the invention before the compound administration,

ii.

the level of fluorescence emission and / or luminescence in another mammal of the invention, which has not been administered the compound; or

iii.

a standard level of fluorescence emission and / or luminescence for mammals according to i or ii;

where a favorable change with the level of indicator (fluorescence and / or luminescence) in relation to the reference change using normal animals not affixed by the tumor induced under comparable conditions, indicates that the compound It is active to treat this disease.

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In a preferred embodiment of this aspect, the Mammalian of the invention is a mouse. In an even more realization Preferred is a nude mouse (nu / nu). In an even more realization Preferred cassette introduction IRES-EGFP-Luciferase is performed 149 nucleotides below the TGA stop codon within the 3'UTR region of the Flt4 gene. And in another even more preferred embodiment the mammal of the invention is homozygous for the introduction of the cassette IRES-EGFP-Luciferase in the region 3'UTR of the Flt4 gene.

Another aspect of the invention relates to a endothelial cell detection and isolation method of lymphatic vessels at different stages of the development of a mammal non-human, comprising:

to)

disintegrate the tissues of said mammal non-human,

b)

detect and isolate the cells that express EGFP by flow cytometry.

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Throughout the description and the claims the word "comprises" and its variants not they intend to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and features of the invention will be partly detached of the description and in part of 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.

Description of the figures

Fig. 1. Generation of the VEGFR3-IRESEGFPluc Kl model: gene targeting strategy .

A) Scheme of the structure of the Flt4 gene, the gene targeting vector used and the allele obtained by homologous recombination in ES cells. The position of the ScaI restriction sites used in the Southern blot analysis of the recombinant clones is also shown, as well as the position of the DNA fragments used as probes in said analysis. Finally, the structure of the recombined allele is shown once the positive selection cassette (PGK-neo) has been removed by Flp recombinase activity.

B) Analysis of recombinant clones by Southern Blot The size of the bands corresponding to WT (Flt4 +) and recombinant alleles (Flt4-neo-IRESEGFPluc)

C) Excision of the PGKneo cassette by Flp recombinase was analyzed by PCR with the indicated primers in figure A to differentiate Flt4-neo-IRESEGFPluc (left) and Flt4IRESEGFPluc (right).

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Fig. 2. Analysis of the expression of VEGFR3 and EGFP in the reporter model .

A) Analysis of VEGFR3 expression in embryos of 13.5 days of development by Northern blot. Be analyzed 15 µg of total RNA of the indicated genotypes using a 500 bp probe specific for messenger RNA from VEGFR3. The size of the messenger RNA is indicated for both the Wild genotype how to knockin. GAPDH levels are Show as load control.

B) Analysis of VEGFR3 expression in embryos of 13.5 days of development by Western Blot. Be analyzed 100 µg of protein extract for each of the genotypes indicated in 10% SDS-PAGE. In the figure Immunoblots for VEGFR3 and EGFP are shown. It is included as Load control the immunoblot of α-Tubulin. It is observed that VEGFR3 expression levels remain constants in different genotypes.

C) Analysis of the expression of VEGFR3-EGFPluc in KI / KI embryos at different stages of development through confocal microscopy. A specific expression of EGFP in the lymphatic vessels of embryos while no expression is detected in the vessels blood, which are recognized by the autofluorescence of the erythrocytes

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Fig. 3. VEGR3 expression monitoring during postnatal development in the reporter model .

Luciferase activity in vivo in females VEGFR3-IRESEGFPluc (n = 8) at the ages of 2, 3, 4, 5, 8, 10 and 15 weeks was monitored in an IVIS Spectrum.

A) Representative images of the activity of Luciferase monitored in the dorsal and ventral areas of each animal.

B) Quantification of luciferase activity shown in the images in A. Luciferase activity is expressed in photons per second per cm2 to correct for the difference in size of animals at different ages. The data They are shown as mean + SD.

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Fig. 4. Analysis of VEGFR3 expression in the reporter model in skin healing trials .

A) Representative images of the issuance of Luciferase during a skin healing trial with or without dexamethasone administration (DEX).

B) Quantification of luciferase emission (B.1) and healing (B.2) during the trial. The data is show as mean + SD.

A maximum of Luciferase emission on day 8 of the trial around wound ends, which correlates with scarring of the 30% Luciferase expression is not detected after day 23 when The wound is completely healed. Treatment with dexamethasone induces a significant decrease in the emission of Luciferase on days 8-10 of healing, without However, the signal is the same in animals treated from day 15, when the wound is healed approximately 40%. He Dexamethasone treatment causes a decrease in macrophage recruitment to the healing zone by altering the process kinetics Keeping this in mind, and that macrophages express VEGFR3, the emission peak that is observed around the day 10 in untreated animals may reflect the process of macrophage recruitment to the wound area. The reduction in macrophage recruitment can also explain the delay in the healing process as shown in Fig. B2.

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Fig. 5. VEGFR3 expression associated with inflammation induced by the contact hypersensitivity test (CHS) .

Mice VEGFR3-IRES-EGFPluc KI / KI (n = 5) is pre-sensitized by topical treatment with Oxazolone in the abdomen. Six days later the inflammation was induced in the left ear of the animals by topical administration of 1% oxazolone. Another group of animals to which CHS was induced (n = 7) was also treated with dexamethasone, administered intraperitoneally at a daily dose of 5 mg / kg of weight.

A) Luciferase emission measured daily in vivo during treatment

B) Quantification of the emission shown in A. The data is shown as mean + SD. (OXA) Oxazolone; (DEX) Dexamethasone.

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Examples

The invention will be illustrated below through tests carried out by the inventors, which puts manifest the specificity and effectiveness of in the monitoring of Lymphangiogenesis / angiogenesis / inflammation processes.

Strategy for the construction of the Knockin model (Fig. 1)

The gene encoding the EGFPluc fusion protein preceded by an IRES sequence ( Internal Ribosome) has been introduced into the 3 'untranslated region (UTR) of the gene encoding the VEGFR3 receptor (flt4), whose expression is specific for endothelial cells. Entry Site ). The IRES-EGFPluc fusion is introduced between the termination codon of the flt4 gene and the polyadenylation sequence. This strategy allows to generate a bicistronic messenger that gives rise to the simultaneous expression of two genes with the same transcriptional regulation and therefore, in this case, to direct the expression of the EGFPluc fusion specifically to the endothelial cells in which the flt4 gene is transcribed. without altering the endogenous expression thereof.

In order to obtain the Flt4-IRES-EGFPluc knockin allele, a "universal" targeting vector was first designed to apply this same strategy to any gene in the murine genome. The structure of this vector is that shown in Figure 1A and is prepared to clone the homology arms of the gene of interest in the unique NotI and SalI sites.

NotI and SalI are unique sites in the vector that recognize these restriction enzymes and where they can be inserted the homology arms of any gene. PGK-neo and PGK-hsvTK are the positive selection markers and negative respectively.

This vector contains a marker that expresses a bacterial neomycin resistance gene under the control of gene promoter encoding mouse phosphoglycerate kinase (PGK-neo). The PGK-neo cassette allows the selection of clones in which the vector has been integrated, either randomly or by homologous recombination. This bookmark is flanked by frt sites for later elimination mediated by Flp recombinase. The vector includes a marker for selection negative that is lost if the vector is integrated by recombination Homologous but not if it is randomly integrated. The latter is the cassette that expresses the herpes virus thymidine kinase gene (hsvTK) also under the control of the PGK promoter. The loss of this cassette is selected in the presence of ganciclovir. These two elements allow you to select the cells that have incorporated the vector by homologous recombination. The inclusion of the marker negative selection provides an increase of 5-10 times in the frequency of homologous recombinant clones.

The vector also contains restriction sites unique to SalI and NotI enzymes that are rare in the mouse genome, and they can be used to clone the arms of homology 5 'and 3' respectively.

The reporter gene that has been used is a EGFP-luciferase fusion protein. EGFP (Enhanced Green Fluorescent Protein) is a variant of the protein GFP, brighter and with better levels of expression in cells mammal. The fusion between the green fluorescent protein and the Luciferase has the advantage that allows the monitoring of its expression both by fluorescence uptake and bioluminescence

Finally, the homology arms used for the construction they have been obtained from genomic DNA of C57BL / 6, and have been amplified by PCR from a BAC (RP23-445K3) that contains this gene. The size of each of the arms is 3.3 Kb and 3.7 Kb the 3 'and 5' respectively. The first ones used for the amplification of 5 'homology arm are:

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Flt45F SEQ ID NO: 3

Flt45R SEQ ID NO: 4

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and for the 3 'arm they are:

Flt43F SEQ ID NO: 5

Flt43R SEQ ID NO 6.

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The amplification was done using the TAQ Takara TAQ LA polymerase by 2 cycles of pre-PCR (98 ° C 10 s, 54 ° C 1 m, 68 ° C 5 m) and 30 cycles PCR (98 ° C 10 s and 68 ° C 5 m).

Once the vector was constructed, it was amplified and after linearizing it by digestion with NotI, it was electroporized in embryonic stem cells (ES) V6.4 of hybrid genetic background F1 [C57BL / 6 129SvJ], typically 17 million cells are electroporated with 20 micrograms of linearized DNA in 0.8 ml of PBS, at 250 volts and 500 microFaradios, in a 0.4 cm wide bucket. The Evaluation of homologous recombination frequency and selection of homologous recombinant clones was performed by analysis by Southern Blot For this, genomic DNA was extracted from 60 clones. For check homologous recombination in the 5 'arm, the DNA is digested with ScaI and analyzed by Southern blot using as probe a 262 bp fragment that hybridizes in an area outside the 5 'homology arm. 3 'arm recombination was analyzed for similar way, digesting genomic DNA with ScaI and using as a probe for Southern Blot a fragment of 324 bp. Of the 60 Clones analyzed, 18 were positive for both arms of homology

Finally two of the recombinant clones (ESIM4.31 and ESIM4.52) were incorporated into developing embryos through the aggregation technique. To do this, morulas were removed of mice of strain CD1, at day 2.5 of pregnancy. These embryos, once the pellucid zone has been removed by digestion with solution from Tyrode, they were added with clusters of about 6-8 embryo cells. The embryos added (approximately 70) are incubated overnight in KSOM medium at 37 ° C, 5% CO2 and daily next they were transferred to the oviduct of pseudogestant females of the strain CD1 a day 0.5 of pseudogestation. The delivery took place at 19 days after embryo transfer. Between the chimeras born pups were distinguished by the presence of pigmentation in the skin and hair, which indicates the contribution of ES cells, since the host embryo comes from a strain albino 10 chimeras were obtained (ESIM4.31, 5 males (1) 100%, 1 (70%), 2 (50%), 1 (30%) and for the clone ESIM4.52, 5 males 100%).

The best male chimeras, crossed with CD1 females to analyze the contribution of ES cells to the germ line (appearance of pigmented offspring). 7 chimeras (of 7 tested) transmitted the pigmentation to their offspring. Of 71 offspring analyzed by PCR, 20 contained the recombinant allele. These animals, heterozygous for the allele Flt4-IRES-EGFPluciferase, se They crossed each other to generate homozygous animals. The frequency of homozygous animals born from these crosses was 25%, indicating that the homozygous mutation does not result in lethality embryonic or perinatal.

Expression analysis by Northern and Western blot (Fig. 2)

For Northern assays, RNA was extracted total from embryos to day of development E13.5 of the indicated genotypes, by extraction with Trizol. Were analyzed 15 micrograms of total RNA of each genotype. As a probe it was used a fragment of 500 bp corresponding to the 3'UTR zone of the flt4 gene external to the reporter's integration site IRES-EGFPluciferase. The probe for GAPDH corresponds to a 400 bp fragment of the gene coding sequence of the Rat GAPDH.

To analyze protein levels by Western blot, 100 micrograms of total protein extract obtained from embryos per day E13.5 of each genotype, they were resolved on a 10% SDS-PAGE gel. Proteins are transferred to a nitrocellulose membrane and hybridized with anti-VEGFR3 antibodies (AFL4 from Becton Dickinson) and anti-EGFP (AB3080 Chemicon International) at dilutions 1:60 and 1: 400 respectively.

EGFP expression monitoring in embryos (Fig. 2)

Embryos on different days of gestation are extracted from the uterus in PBS and observed under a microscope confocal Leica TCS-SP5 (AOBS) equipped with the LAS AF acquisition software. To excite the EGFP a 488 nm argon laser.

Determination of luciferase activity in extracts

Luciferase activity in extracts of Different organs and tissues were measured using a commercial kit of Promise (E-1501) following the recommendations of the maker.

Determination of luciferase expression in vivo . (Fig. 3, 4 and 5)

Animals at different ages and of different Genotypes are anesthetized by isofluorane inhalation. TO They are then given D-luciferin (Xenogen XR-1001) at a concentration of 15 mg / ml in PBS by a single intraperitoneal injection. The luciferin dose It is 150 mg / kg of weight. Injected animals are observed in a IVIS Spectrum (Xenogen Corp.) acquiring images for 30 seconds continuously until the maximum of issuance (approximately 20 minutes after administration of the Luciferin).

Skin healing trials. (Fig. 4)

In this test, females 11-14 weeks old and heterozygous for the VEGFR3-EGFPluciferase Kl allele are used. Animals are anesthetized by isofluorane inhalation. The dorsal area is shaved and a circular wound is made in the skin, approximately at an average height of the dorsal area, with an 8 mm diameter biopsy punch, cleaving the skin and the fleshy panicle. At different days the emission of luciferase in an IVIS Spectrum is measured as described above. In this case, the ROI ( region of interest ), where the signal is measured, corresponds to the initial wound area for each mouse at each point. In parallel, the diameter of the wound is measured, in vertical (V) and horizontal (H) orientation with a caliber. To calculate the area at each point, the means V x H are multiplied. Healing is represented at each point as the percentage size of the wound with respect to the size of the wound.
day 0.

To study the contribution of inflammation to the healing process, in the same trial some animals were treated with dexamethasone (Sigma D1756) at a concentration of 1 mg / ml in Ethanol and at a dose of 5 mg / kg of administered weight by daily intraperioteneal injection. Control animals They were treated in the same way with vehicle only.

Contact hypersensitivity test (Fig. 5)

Homozygous females for the allele VEGFR3-EGFPluciferase Kl of approximately 11 weeks of age were pre-sensitized by topical administration of 50 microliters of 2% oxazolone in acetone / oil 4: 1 v / v in the abdomen previously shaved. Six days later (day 0) inflammation was induced by administration Topical 10 microliters of 1% oxazolone in the same vehicle in The animal's left ear. The right ear was treated only with vehicle as internal control. In parallel, another group of animals of the same age and genotype were treated with dexamethasone as described above. Luciferase emission was measured daily at day 0 and for the next six days at the beginning of treatment using the IVIS Spectrum as described in the previous sections. In this case the ROI is just the treated ear or The ear control of each animal.

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13

Claims (13)

1. Genetically modified non-human mammal whose cells express the construction IRES-EGFP-Luciferase in the region 3'UTR of the Flt4 gene with the same pattern of receptor expression VEGFR-3, both in space and time. 2. Genetically modified non-human mammal according to the preceding claim, wherein the expression of the construction IRES-EGFP-Luciferase se achieved by entering SEQ ID NO: 1 in the animal or One of his ancestors. 3. Genetically modified non-human mammal according to any of claims 1-2, wherein The mammal is immunosuppressed. 4. Genetically modified non-human mammal according to any of claims 1-3, wherein cassette introduction IRES-EGFP-Luciferase is performed 149 nucleotides below the TGA stop codon within the 3'UTR region of the Flt4 gene. 5. Genetically modified non-human mammal according to any of claims 1-4, which is homozygous for the introduction of the IRES-EGFP-Luciferase cassette into the 3'UTR region of the Flt4 gene. 6. Genetically modified non-human mammal according to any of claims 1-5, wherein The mammal is a mouse. 7. Gene-targeting vector comprising the genetic construct with the cDNA encoding the EGFP-luciferase fusion protein, preceded by an internal ribosome entry sequence (IRES), the genomic sequences of the Flt4 locus flanking the point of integration of the IRES-EGFP-luciferase construct in the 3'UTR region of the Flt4 gene, and a positive selection cassette . 8. Gene-targeting vector according to the preceding claim wherein the positive selection cassette comprises the promoter of the mouse phosphoglycerate kinase gene, a neomycin resistance gene and a SV40 virus polyA signal, flanked by frt sequences. 9. Vector according to any of claims 7-8, further accompanied by a negative selection cassette comprising, the promoter of the mouse phosphoglycerate kinase gene, the coding sequence of the herpes simplex virus thymidine kinase gene, and a polyA signal of SV40 virus. 10. Method of genetic modification for build a genetically modified non-human mammal according to any of claims 1-6, which understands:

to.

build a vector according to any of claims 7-9.

b.

transfect said construction in a population of non-human embryonic stem cells, and select embryonic stem cells that have stably integrated the vector of (a) in its genome by homologous recombination.

C.

incorporate the stem cell clones embryonic recombinants from step (b) to non-host embryos humans so that recombined cells contribute to all the cell lineages of the embryo.

d.

allow the development of these embryos to obtain a chimeric non-human mammal with the allele that expresses the construction IRES-EGFP-Luciferase in the region 3'UTR of the Flt4 gene.

and.

cross the chimeric non-human mammal of step (d) to produce heterozygous non-human mammals for the modification introduced, and optionally,

F.

cross heterozygous individuals according to (e) each other to produce a genetically non-human mammal homozygous modified for construction expression IRES-EGFP-Luciferase in the region 3'UTR of the Flt4 gene.

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11. Method according to claim 10, wherein embryonic stem cells are hybrid genetic background V6.4 F1 (C57BL / 6 x 129). 12. Isolated mammalian cell according to any of claims 1-6, or which has integrated by homologous recombination a vector according to any of claims 7-9. 13. Cell line derived from an isolated cell according to claim 12.