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  • A01K2267/0325—Animal model for autoimmune diseases

Definitions

• the invention relates to a non-human transgenic animal in which the expression of the gene coding for insulin is suppressed.
• the single human insulin gene, the two rat insulin genes and the two mouse insulin genes have been cloned for several years.
• mice as in rats the two genes coding for very similar functional insulins (insulins 1 and 2 differ only by two amino acids) produced in similar proportions.
• the sequence of these genes is known, it is considered identical in all the lines of mice. This is not true of the nucleotide sequences of the regions of DNA flanking the gene on either side. To increase the probability of recombination at the site of an endogenous gene, which is very low, the flanking sequences must be most strictly homologous on the recombination vector to be constructed and on cellular DNA. However, this homology is only strict within an inbred line of mice.
• insulin a peptide hormone secreted by ⁇ cells from pancreatic islets, acts through a membrane receptor with tyrosine kinase activity and that the signal it transmits thus plays a fundamental role in the metabolism of carbohydrates. , lipids and proteins. Its deficiency, due to the autoimmune destruction of ⁇ cells during so-called "type I" diabetes, is rapidly lethal, following profound metabolic disorders.
• the three major target tissues for insulin are the liver, adipose tissue and muscle.
• Insulin receptors are present on all cell types in the body and the effect of insulin on these cultured cells, other than the three types mentioned above, is admitted, which can induce DNA synthesis, translation of messenger RNAs and assimilation of glucose.
• IGFs insulin-like growth factors
• Each of the ligands can also bind the receptor of the other ligand, but its possible functional significance is unknown. The fact remains that the absence of insulin in children and adults has the disastrous effects mentioned above, mainly due to liver failure.
• no example is known in humans or in animals of mutations which abolish insulin synthesis (null mutation) and, therefore, the possible role played by insulin in the embryo is unknown. His development.
• One of the aspects of the invention is to provide an experimental model enabling the screening of drugs active on pathologies involving insulin.
• One aspect of the invention is to provide transgenic mammalian animals in which at least one of the genes coding for insulin is no longer expressed.
• One aspect of the invention is to provide transgenic mammalian animals in which the gene encoding insulin 1 is no longer expressed.
• One aspect of the invention is to provide transgenic mammalian animals in which the gene encoding insulin 2 is no longer expressed.
• One aspect of the invention is to have the restriction map of insulin genes, which allows recloning fragments of insulin genes and their flanking regions from the line of 129 mice, since embryonic stem cells (ES) come from the same line of mice 129.
• ES embryonic stem cells
• the subject of the invention is the use of a non-human transgenic mammal animal, in which at least one of the alleles of one at least of the two genes coding for endogenous insulin is rendered functionally inoperative with respect to vis the expression of insulin, for the determination of active drugs, on pathologies involving insulin.
• the invention relates to the use of a non-human transgenic mammalian animal in which the expression of endogenous insulin 1 and / or endogenous insulin 2 is suppressed compared to normal expression , especially in the cells of the pancreas.
• insulin denotes the insulin 1 gene and
• mice includes all mammals except humans, advantageously rodents and in particular mice.
• transgenic animal an animal into the genome of which an exogenous gene construction has been introduced, which is inserted either randomly into a chromosome, or very precisely at the locus of an endogenous gene, which results in this last case to the impossibility of the expression of this endogenous gene because it is either interrupted, or replaced entirely or partially by a construction such that it no longer allows the expression of the endogenous gene or a construction which, in addition to the deletion of the endogenous gene, introduces an exogenous gene.
• Such animals are designated as "knock-out” animals or animals whose abovementioned endogenous gene is invalidated.
• the normal expression of the insulin gene can be defined as follows: 1) Determination of the messenger RNA corresponding to one of the genes coding for insulin. This is possible by a retrotranscription of the messenger RNA from an identical primer for the two messengers insulin 1 and insulin 2, followed by the amplification according to a geometric progression of the complementary DNAs generated, by the action of a polymerase. (polymerase chain reaction by reverse transcriptase: RT-PCR), the two homologous and amplified fragments then being separated by electrophoresis, thanks to a Mspl restriction polymorphism which makes it possible to generate a fragment of 71 base pairs for insulin 1 and 112 base pairs for insulin 2.
• the common primers used for "RT-PCR" are 5'-GGCTTCTTTCTACACACCCA-3 'and 5'- CAGTAGTTCTCCAGCTGGTA-3', and the probe common to the two products is an oligonucleotide 5'-ACAATGCCACGCTTCTG -3 '.
• the absence of expression of the insulin 1 and / or 2 gene can be determined as follows:
• mice where the gene coding for insulin has been replaced by a construct such that it can no longer be expressed are first characterized with respect to DNA by analysis with the DNA transfer method (Southern blot ), using a probe consisting of a fragment containing all or part of the region of the gene coding for insulin, this probe being defined in the examples.
• Hybridization of this probe clearly shows that the DNA band corresponding to one of the wild type insulins (that is to say normally present in animals) is no longer present in animals homozygous towards - screw of the mutation, but replaced by a band corresponding to the modified genome.
• RNAs extracted from the tissues expressing at least one of the insulins shows that there is no longer any expression of RNA corresponding to the wild-type gene.
• the primers and the probe used were defined above, in the paragraph entitled "Determination of the messenger RNA corresponding to one of the genes coding for insulin”.
• the cells in which the expression of a gene coding for one of the insulins is suppressed are essentially the cells of the pancreas.
• the suppression of the expression of the insulin 2 gene also occurs in the other tissues where it is expressed, in particular the yolk sac, the brain and the thymus.
• the invention relates more particularly to a non-human transgenic mammal animal, or mammalian cells, in which at least one of the alleles of one at least of the two genes coding for endogenous insulin is rendered functionally inoperative with respect to vis the expression of insulin.
• the animals of the invention are such that one of the two alleles of the gene coding for insulin 1 is made functionally inoperative with respect to the expression of insulin 1.
• the animals of the invention are such that the two alleles of the insulin 1 gene are made functionally inoperative with respect to the expression of insulin 1. In other words , there is no longer any expression of insulin 1. In this case, the overexpression of the insulin 2 gene partially counterbalances the absence of expression of insulin 1; animals live and reproduce, however, without apparent pathology.
• the animals of the invention are such that one of the two alleles of the gene coding for insulin 2 is made functionally inoperative with respect to the expression of insulin 2.
• the animals of the invention are such that the two alleles of the insulin 2 gene are made functionally inoperative with respect to the expression of insulin 2. In other words , there is no longer any expression of insulin 2.
• the overexpression of the insulin 1 gene compensates for the absence of expression of the insulin 2 gene; animals can have transient diabetes mellitus at birth, which disappears after a few days, but they live and reproduce without apparent pathology.
• the animals are such that one of the two alleles of the gene coding for insulin 1 is made functionally inoperative with respect to the expression of insulin 1 and the two alleles of the gene of insulin 2 are rendered functionally inoperative with respect to the expression of insulin 2.
• the animals are such that one of the two alleles of the gene coding for insulin 2 is made functionally inoperative with respect to the expression of insulin 2 and the two alleles of the gene of insulin 1 are rendered functionally ineffective with respect to the expression of insulin 1.
• animals may have transient diabetes mellitus at birth, which disappears after a few days, but they live and reproduce without apparent pathology.
• the animals of the invention are such that the two alleles respectively of the insulin 1 gene and the insulin 2 gene are made functionally inoperative. In this case there is no production of insulin.
• Animals have diabetes mellitus with ketoacidosis which develops from the start of feeding and is lethal in 2 to 3 days. This diabetes is sensitive to the administration of insulin. These animals are designated by the term double nullizygotes.
• the invention relates in particular to a non-human mammal animal or mammalian cells in which at least one of the alleles coding for insulin 1 and / or insulin 2 is replaced by a gene capable of coding for a protein exhibiting an activity enzymatic.
• genes capable of coding for a protein exhibiting activity enzymatic there may be mentioned those of ⁇ galactosidase, of luciferase, of chloramphenicol acetyltransferase, of an aminoglycosylphosphotransferase.
• the gene exhibiting enzymatic activity can be either under the control of the promoter of the insulin gene, or under the control of its own promoter.
• the gene capable of coding for an enzymatic protein replaces at least one of the alleles of the insulin 2 gene.
• the gene capable of coding for an enzymatic protein replaces at least one of the alleles of the insulin gene 1.
• the invention relates to a mammalian animal non-human transgenic or mammalian cells in which the expression of the endogenous gene coding for insulin 1 and that of the endogenous gene coding for insulin 2 are suppressed and containing a transgene allowing the expression of human insulin.
• the invention also relates to the use of a transgenic mammalian animal as defined in the preceding paragraph.
• the transgene allowing the expression of exogenous human insulin corresponds to a fragment of human DNA containing the transcription unit of the insulin gene and its flanking regions, in particular an 11 kilobase fragment.
• the invention relates to a transgenic non-human mammalian animal or mammalian cells, in particular ⁇ , in which the expression of the endogenous gene coding for insulin 1 and that of the endogenous gene coding for insulin 2 are deleted and further contain a transgene allowing expression of human insulin.
• the insulin produced and stored in the ⁇ cells of the pancreas of these animals is exclusively human insulin; the animals are not diabetic, they live and reproduce without apparent pathology and there are genetically stable lines.
• the invention relates to a non-human transgenic mammalian animal or mammalian cells, in particular ⁇ , in which the expression of the endogenous gene coding for insulin 1 and that of the endogenous gene coding for insulin 2 are deleted and additionally containing a transgene comprising the coding sequence of the T antigen of the SV40 virus under the control of the promoter of the rat insulin 2 gene, and allowing the induction of proliferation of ⁇ cells.
• the invention relates to a non-human transgenic mammalian animal or mammalian cells, in particular ⁇ , in which the expression of the endogenous gene coding for insulin 1 and that of the gene endogenous coding for insulin 2 are deleted and further containing on the one hand a transgene allowing the expression of human insulin and, on the other hand, a transgene comprising the coding sequence of the T antigen of the SV40 virus under the control of the promoter of the rat insulin 2 gene previously modified so as to induce the arrest of its transcription under the effect of the presence of tetracycline, and allowing the arrest of the proliferation of ⁇ cells in the presence of tetracycline.
• the invention relates to a non-human transgenic mammalian animal or mammalian cells, in particular ⁇ , in which the expression of the endogenous gene coding for insulin 1 and that of the endogenous gene coding for insulin 2 are deleted and additionally containing on the one hand a transgene allowing the expression of human insulin and, on the other hand, a construct containing the transgene comprising the coding sequence of the T antigen of the SV40 virus under the control of the promoter of the rat insulin 2 gene previously modified so as to induce its transcription under the effect of the presence of a substance, in particular an antibiotic, a hormone, a cytokine or a growth factor , and allowing the induction of proliferation of ⁇ cells in the presence of the above substance.
• a substance in particular an antibiotic, a hormone, a cytokine or a growth factor
• the invention also relates to cells encapsulated in an inert material capable of grafting these cells in vivo under conditions of shelter from the immune system.
• “Inert material” means a substance or a physico-chemical complex which does not modify the grafted biological material and does not induce an immunological reaction of the host, which makes this material suitable for grafting.
• immune system shelter condition means that living transplanted cells are separated from the host immune system so that immune system cells, antibodies, and other rejection factors , cannot reach them.
• the invention also relates to a non-human mammalian animal or mammalian cells, resulting from the crossing of the animals defined above, or from the crossing of any of the animals defined above with another animal of the same species.
• the invention also relates to cells grown from the transgenic non-human mammalian animals described above.
• the invention relates to cell cultures containing one of the above transgenic constructs.
• the invention also relates to a non-human transgenic mammal as obtained by introduction into a blastocyte of embryonic stem cells (ES cells) comprising in their genome one of the above-mentioned transgenic constructions, obtained by homologous recombination, selection of chimeric animals according to a criterion corresponding to the ES line, crossing of selected animals with mice, in particular C57 Black 6 mice, to obtain animals heterozygous with respect to one of the above-mentioned constructions and possibly crossing of two heterozygotes to obtain an animal homozygous with respect to to one of the above constructions.
• ES cells embryonic stem cells
• the homozygote has a genetic background 129 / C57 Black 6 50/50.
• C57 Black 6 mice are advantageously chosen, because this genetic background is more favorable for certain behavioral experiments.
• the invention also relates to a transgenic mammal as produced by crossing transgenic animals expressing one of the transgenic constructs defined above.
• the invention also relates to the process for obtaining a transgenic model for the study of pathologies involving insulin and their treatment comprising:
• the vector used to make the recombination vector being derived from pKS + , containing the neo genes (BamHI fragment from pMCl POLA -Stratagen) at the BamHI and tk site (fragment XhoI / HindIII from pMCtk described in Liu et al. , Cell, 1993, Vol. 75, 59-72) between the Xhol and HindIII sites and designated by pNTK,
• mice in particular C57BL / 6 mice giving heterozygous animals with respect to one of the constructs as defined above, and
• the invention also relates to a method for screening for drugs active on pathologies involving insulin, in particular diabetes, comprising the administration of a drug to be tested to a transgenic non-human mammal or to cells of transgenic non-human mammals
• the invention also relates to a transgenic construct in which:
• ES embryonic mouse strains
• the vector used to make the recombination vector being derived from pKS + , containing the neo genes (BamHI fragment from pMCl POLA -Stratagen) at the BamHI and tk site (XhoI / HindIII fragment from pMCtk described in Liu et al., Cell, 1993, Vol. 75, 59-72) between the Xhol and HindIII sites and designated by pNTK,
• the invention also relates to the genomic DNA of insulin 1 of the inbred mouse line 129, characterized by the following restriction sites, by reference to the origin of the transcript located at position + 1:
• the invention also relates to the genomic DNA of insulin 2 from the inbred mouse line 129, characterized by the following restriction sites, by reference to the origin of the transcript located at position + 1:
• mutant mice of the invention in the study of diabetes, compared to already existing animal models, such as the mouse of the NOD line or the rat of the BB line, is that the absence of insulin is total from birth, that this absence exists from embryonic life, that it exists without the autoimmune disease, cause of the disease of the aforementioned animals and of type I human diabetes. They make it possible to explore the proper role of absence insulin with no associated immune component.
• mice in which only one, two or three insulin genes are disabled have no detectable chronic diabetic syndrome.
• the mutant mice of the invention make it possible to determine whether, at an advanced age, these animals do not develop vascular complications, usual in the diabetic even in appearance apparently well balanced by insulin therapy. These complications are responsible for blindness, kidney failure and arteritis. It is possible that the mice defined above are poorly balanced so far imperceptibly but that they could represent an animal model for the vascular complications of diabetes, a model which does not exist at all at present.
• the mouse is currently the only animal from which it is known to derive lines of ⁇ pancreatic cells in a reproducible manner. In addition, these cells retain their essential functional properties, synthesis and storage of insulin, and glucose-induced secretion.
• the possibility of deriving such cells from mutant animals lacking insulin synthesis is an important opening with a view to developing ⁇ cells capable of being used in cell therapy for diabetes. Indeed, the introduction of a human insulin transgene into mutant mice or even directly into ⁇ cells already established in culture, makes it possible to obtain ⁇ cells which can be grafted on to humans (thanks to the methods of cell encapsulation, which protects the grafted cells of the host's immune system) which secrete under the physiological control of the host from human insulin, i.e. a self-protein which does not induce itself immune reaction.
• Figure 1 It represents the targeted interruption of Insl ( Figure la) and Ins2 ( Figure lb).
• FIG. 1a The structures of the targeting vectors, as well as the wild type (wt) and the recombinant alleles are represented respectively in FIG. 1a for Ins1 and in FIG. 1b for Ins2. Restriction enzymes and probes used for genotyping mouse and cell DNA have been identified.
• Figure 2 RT-PCR analysis of Ins1 / Ins2 expression in the pancreas of wild mice and nullizygous double mutants.
• the ⁇ -actin mRNA is amplified as a control.
• Figure 5 Restriction map of the insulin 1 gene of the line of inbred mice 129.
• Figure 6 Restriction map of the insulin 2 gene of the line of inbred mice 129.
• the null mutation of the insulin 1 gene (Ins1) or that of the insulin 2 gene (Ins2) required the following steps:
• mice carrying a null mutation of the two alleles of the Insl gene and of the lns2 gene are obtained in two stages.
• crossing a nuUizygote mouse for Insl with a nuUizygote mouse for Ins2 produces a first generation of mice all heterozygous for each of the mutations.
• the crossing of the latter between them generates with a frequency of 0.0625 nullizygous double mice (i.e. on average one mouse on
• cDNAs complementary DNAs corresponding to one, one to Ins1, the other to Ins2, labeled with
• a library of 129 mice available on the market (Stratagene) made it possible to clone several ⁇ Ins2 phages.
• a - For Insl a Hind III / SmaI fragment of 8.7 kilobases (kb) and an Apal / Xhol fragment of 8.2 kb (corresponding to the flanking regions upstream (in 5 ') and downstream (in 3') Ins1) were separately subcloned into a plasmid pSK + previously digested with Hind III and SmaI, for one, Apal and Xhol, for the other, resulting in plasmids p34 and p4.
• Another BamHI / HindIII fragment (corresponding to the 5 'region of InsI) was also cloned into pSK +, giving pR1.
• the vector used to make the recombination vector is derived from pKS + containing the neo genes at the BamHI and tk site between the Xhol and
• LacZ encodes the enzyme ⁇ galactosidase which splits lactose into glucose and galactose.
• the use for substrate of the enzyme of a colorless X-gal precursor generates a product of intense blue coloring which can be visualized on anatomical or histological preparations and titrated by colorimetry.
• the pKS + derivative defined above was first modified by inserting an Nsil linker at the HindIII site, giving plO.
• Synthetic probe 3 corresponds to the XhoI / EcoRI fragment of pl2 and probe 4 to the neo fragment (see FIG. 2).
• the ES cell cultures and the manipulation of mouse embryos were carried out according to the methods described [2-4].
• the DNAs of the recombination vectors were linearized by Notl digestion before being electroporated in ES cells of the D3 line.
• 3 recombinant clones for Insl and 12 for Ins2 were identified by molecular analysis (see below).
• Ten to fifteen cells from these clones were micro-injected into the cavity of blastocysts of the C57BL / 6 mouse line (B6) which were then transferred into the oviduct of pseudogestant females.
• B6 C57BL / 6 mouse line
• Two independent cell clones for each of the mutations were used.
• First generation mice with thick coats are derived from germ cells derived from ES cells. Molecular analysis (see below) makes it possible to identify those of them which have inherited the recombined Ins allele, that is to say the null allele.
• mice heterozygous for a mutation were crossed between them.
• a quarter of nullizygous animals were found, that is to say homozygous for the considered null mutation.
• the crossing between them of these nullizygote animals made it possible to establish nullizygote lines either for the Insl gene or for the Ins2 gene.
• the presence of the null mutation in recombinant ES cells, to the exclusion of any other random insertion of the recombination vector is detected by DNA analysis.
• the restriction fragments generated from DNA are separated by electrophoresis, transferred to a nylon membrane on which they are incubated with a specific radioactive probe, including hybridization to DNA fragments of determined size, revealed by autoradiography. , allows to conclude that the expected mutation is present.
• the mutation introduced into the Ins2 gene comprises the coding sequence of the LacZ gene which is placed under the control of the regulatory sequences of the In 2 gene.
• the LacZ gene now functions like the Ins2 gene and in its place.
• the product of this gene, the ⁇ galactosidase enzyme is capable of cleaving a colorless precursor by generating a blue derivative. This can be seen by transparency on an entire pancreas, on histological sections. It can be highlighted and its concentration measured by colorimetry of cellular or tissue extracts.
• the insulin-free transgenic mice of the invention make it possible to develop or test strategies for alternative diabetes therapies.
• mice whose only insulin produced is human insulin 1) Obtaining mice whose only insulin produced is human insulin:
• Transgenic mouse lines expressing the human insulin gene are available. These mice are obtained by micro-injecting into the pronucleus of zygotes (oocyte fertilized by a spermatozoon and in which the two male and female pronuclei are always distinct) a fragment of human DNA of 11 kilobases containing the insulin gene (1430 base pairs). Some of the mice derived from the development of these embryos have integrated foreign DNA (the transgene) into one of their chromosomes. They express the transgene, have a fraction of their insulin which is human insulin and have no pathology, their overall amount of insulin synthesized and stored being normal. They were used to establish, by crossing, lines of transgenic mice for the human insulin gene. One of these lines (Tgl71), in which the human transgene is integrated into chromosome No. 18 [6] is used to introduce the human transgene into a line devoid of functional mouse insulin genes.
• Tgl71 in which the human transgene is integrated into chromosome No. 18 [6
• mice were crossed with mice carrying null alleles of Insl and Ins2.
• First generation mice carrying a null allele for Ins1, a null allele for Ins2 and a human transgene were obtained. Crossing them will make it possible to obtain individuals carrying two null alleles of Insl, two null alleles of Ins2 and two alleles of the transgene. Unlike double nullizygous mice, these mice survive the neonatal period because they produce insulin. This insulin is only human insulin.
• mice for a gene construct comprising the coding sequence of the T antigen of the SV40 virus under the control of the promoter of the rat Ins2 gene (Ins-Tag transgene). These mice express the T antigen in ⁇ cells, which has the effect of inducing their proliferation, both in vivo and when they are cultured. This notable proliferation in culture, which does not exist for ordinary mouse ⁇ cells, makes it possible to establish ⁇ cell lines, retaining their essential biological properties. It is however impossible to stop their proliferation, which remains uncontrolled. [7-9], More recently, a line of mice carrying the same Ins-Tag transgene has been constructed, previously modified so as to induce the stopping of its transcription under the effect of the administration of tetracycline.
• ⁇ cells in culture derived from such animals are stopped by the addition of tetracycline to the culture medium [10].
• Other transgenic mice can be constructed, in which the Ins-Tag transgene is modified so as to induce transcription under the effect of a drug.
• the proliferation of ⁇ cells, both after birth and in culture, is only possible after administration of this drug and ceases with it. This case is much more operationally interesting to consider cell therapy.
• transgene encoding the T antigen in one of the three forms described above, into the genetic heritage of mice producing only human insulin, makes it possible to establish different lines of ⁇ cells capable of being transplanted into a host.
• mouse ⁇ cells producing only human insulin makes it possible to test the effectiveness of this cellular treatment of mouse or rat diabetes.
• Such cells especially if their proliferation in vivo is dependent on the administration of a drug, are likely to be used in clinical trials.
• Another avenue of cell therapy is represented by attempts to modify fibroblasts or other cell types other than ⁇ cells so that they secrete insulin in a glucose-regulated manner. Insulin-free mutant mice can be used to test the effectiveness of these treatments.
• mice carrying a mutation in an In 2 gene have the LacZ gene which is induced and active as is the Ins2 gene.
• the introduction of the transgene coding for the T Ins-Tag antigen in these mice, by the crosses of suitable mice, makes it possible to develop from these animals one or more ⁇ cell lines whose ⁇ galactosidase activity serves as an indicator for to screen drugs of all kinds likely to induce or on the contrary to repress the transcription of the Ins 2 gene.
• double nullizygous mice for Ins and expressing the human insulin gene can be used to assess the possible immunogenic effect of synthetic insulins or of recombinant insulins which may be used in humans for treatment of diabetes or other indications.
• mice nullizygous for the Ins2 gene do not have obvious blood sugar disorders. However, at birth, some of them have a transient glycosuria of a few hours. This observation is explained by the fact that before birth, the transcript level of the other gene, Insl, is similar to that of non-mutant mice, but that, after birth, there is a compensation for l absence of Ins2 by the increased transcriptional activity of Insl. On the other hand, this massive transcriptional compensation was not found on the part of the Ins2 gene when it is a case of mice nullizygous for Insl.
• the transient diabetes observed in newborn mice nullizygous for Ins2 has been observed, for a longer period (a few days) in some of the animals having three copies of disabled Ins genes. These mutant animals are examined for the presence of vascular abnormalities. There are in fact no model animals at present for vascular complications of diabetes mellitus and the mutants which are the subject of this invention may be a first example.
• Study of the role of insulin in autoimmunity In the normal state, the Ins2 gene, and not the Insl gene, is transcribed in the thymus of mice. The same phenomenon has been described for the unique insulin gene in humans.
• nullizygous mice for Ins2 offers the opportunity to examine the role of expression of Ins2 in the thymus in the normal establishment of autoimmunity.
• the analysis of this question requires the use of hybridomas T particular, established in other lines of mice and usable only in these lines.
• the introduction of the nuUizygotie for Ins2 into one of these lines will be carried out by suitable crosses.
• a nuUizygote line for Ins 2 which can be analyzed with the available hybridomas.
• mice in which all the insulin produced is human insulin. These mice were obtained by introducing a human insulin transgene into a line of mice whose Insl and Ins2 genes were invalidated by homologous recombination.
• the human transgene is an 11 kilobase fragment of genomic DNA comprising the transcription unit of the human (pro) insulin (Insh) gene, surrounded by an upstream DNA fragment (5 ′ fragment) 4 kilobases and a downstream fragment (3 'fragment) of approximately 5.5 kilobases.
• the functional activity of the Insh transgene was characterized by the detection and determination of human peptide C in the urine by a radio immunological test (RI A), the detection of a human insulin transcript in the preparations d Total RNA of the pancreas and the demonstration of the initiation of this transcript at the physiological site, the demonstration that the human protein produced is only found in the ⁇ cells of the endocrine pancreas by immunocytofluorescence using specific antibodies, the demonstration that it is co-located with mouse insulins in the same secretory granules of these ⁇ cells.
• RI A radio immunological test
• the transgene was located on chromosome 18 of the mouse. Quantification established that human insulin represents 47% of the total insulin synthesized and stored in the transgenic islets.
• the human transgene was placed on a C57B1 / 6 inbred genetic background by a series of 12 backcross crosses with C57B1 / 6 animals.
• Insh transgenic mice were crossed with mice described above, one copy of the Ins2 gene and the two copies of the Insl gene are invalidated.
• individuals identified by DNA analysis were identified as having a copy of Insl and a copy of Ins2 disabled and a copy of the human transgene.
• They can serve as starting material for developing cell lines of ⁇ cells of which all the insulin produced is human insulin.
• mice can also be used for crosses with non-mutant mice to recover individuals carrying the Insl, Ins2 mutation or the human transgene in the heterozygous state, the individuals of each of the three types being able to be crossed separately with each other to obtain again three stable and viable homozygous mouse lines for each of the mutations or for the transgene.

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