WO2004063381A1 - Targeted transgenesis using the rosa26 locus - Google Patents
Targeted transgenesis using the rosa26 locus Download PDFInfo
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- WO2004063381A1 WO2004063381A1 PCT/EP2004/000065 EP2004000065W WO2004063381A1 WO 2004063381 A1 WO2004063381 A1 WO 2004063381A1 EP 2004000065 W EP2004000065 W EP 2004000065W WO 2004063381 A1 WO2004063381 A1 WO 2004063381A1
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- Prior art keywords
- cells
- rosa26 locus
- promoter
- gene
- recombination
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Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/027—New or modified breeds of vertebrates
- A01K67/0275—Genetically modified vertebrates, e.g. transgenic
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
- C12N15/90—Stable introduction of foreign DNA into chromosome
- C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
- C12N15/907—Stable introduction of foreign DNA into chromosome using homologous recombination in mammalian cells
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2217/00—Genetically modified animals
- A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2217/00—Genetically modified animals
- A01K2217/07—Animals genetically altered by homologous recombination
- A01K2217/072—Animals genetically altered by homologous recombination maintaining or altering function, i.e. knock in
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2217/00—Genetically modified animals
- A01K2217/07—Animals genetically altered by homologous recombination
- A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2800/00—Nucleic acids vectors
- C12N2800/30—Vector systems comprising sequences for excision in presence of a recombinase, e.g. loxP or FRT
Definitions
- the invention provides a method for targeted transgenesis using the Rosa26 locus. Suitable nucleotide acid sequences and vectors for the targeted transgenesis and recombinase mediated transgenesis are provided.
- the Rosa26 locus proved to be a suitable integration site allowing strong and predictable expression of inserted transgenes carrying exogenous promoters.
- transgenic mice by nuclear injection of purified DNA into fertilized eggs is a widely used approach for studying gene or promoter function in vivo.
- the level and pattern of expression often varies strongly depending on copy number, configuration, and integration site of the transgene.
- founder mice occasionally do not transmit the transgene.
- a number of different founders need to be generated and tested in order to identify a useful strain, which is a laborious and time-consuming undertaking (Bradley et. al., Nature Genet., 14:121-123 (1996); Jasin et al., Proc. Natl. Acad. Sci.
- eNOS promoter-LacZ reporter gene placed in the Hprt locus was found to be inactive in hepatic vessels that otherwise express the endogenous eNOS gene (Guillot et al., Physiol. Genomics, Mar. 13, (2):77-83 (2000).
- HPRT gene is on the X chromosome, transgene expression at this locus is subjected to random X-inactivation. The expression of the transgene in all cells of the female, therefore, requires the generation of homozygotes.
- the rosa26 locus had been identified by random insertion of retroviral sequences and a ⁇ -galactosidase-neomycin resistance fusion gene into the genome of mouse embryonic stem cells (Zambrowicz et al., Proc. Natl. Acad. Sci. USA, 94, 3789-94 (1997)).
- the rosa26 promoter appeared to mediate ubiquitous expression of promoter-less genes both in embryos and adult mice (Kisseberth et al., Dev. Biol., 214:128-138 (1999); Zambrowicz et al., Proc. Natl. Acad. Sci. USA, 94, 3789-94 (1997)), albeit at different levels in different organs (Vooijs et al., EMBO reports, 21:292-297 (2001)).
- WO 99/53017 describes a process for making transgenic animals which ubiquitously express a heterlogous gene, wherein the heterologous gene is under the control of a ubiquitously expressed endogenous promoter, e.g. that of the mouse Rosa26 locus.
- a ubiquitously expressed endogenous promoter e.g. that of the mouse Rosa26 locus.
- R. Dacquin et al., Dev. Dynamics 224 :245-251 (2002) and K. A. Moses et al., Genesis 31: 176-180 (2001) utilize the transgenic mouse strain R26R obtained according to WO 99/53017 for the expression of heterlogous genes.
- WO 02/098217 describes a method of targeting promoter-less selection cassettes into transcriptionally active loci, such as the Rosa26 locus.
- WO 03/020743 (published March 13, 2003) describes the expression of transgenes in vivo by targeting protected transgene cassettes into predetermined loci (e.g. the Rosa26 locus), such that the introduced tissue specific exogenous promoter has at least . some tissue specific activity.
- the protected transgene cassette contains (from 5' to 3' direction) a transcriptional stop signal, the exogenous tissue specific promoter and the gene of interest.
- the presence of a transcriptional stop signal is vital for the method of WO 03/020743 as therewith the expression pattern is determined primarily by the nature of the tissue specific exogenous promoter.
- the present invention is based on the finding that a particular chromosomal locus present within the eukaryotic genome (including that of mammalian ES cells), namely Rosa26, supports the preservation of the inherent activity of heterologous promoters inserted through homologous recombination at that locus.
- This chromosomal locus is therefore useful in the context of the "targeted transgenesis" approach for the efficient generation of transgenic organisms (such as mice) with a predictable transgene expression pattern.
- Such a “targeted transgenesis” method comprises consecutive experimental steps.
- a gene expression cassette comprising a suitable promoter (e.g. a ubiquitous or tissue specific promoter, either inducible or constitutive) functionally linked to a gene of interest has to be created; subsequently a vector for the targeted insertion of the above mentioned gene expression cassette into the Rosa26 locus has to be generated; the insertion of the above mentioned gene expression cassette into the Rosa26 locus through homologous recombination or site specific recombination in embryonic stem cells follows; finally transgenic mice are generated by the injection of such genetically modified ES cells into blastocysts.
- a suitable promoter e.g. a ubiquitous or tissue specific promoter, either inducible or constitutive
- step (a) introducing a functional DNA sequence into the Rosa26 locus of starting eukaryotic cells, preferably said functional DNA sequence is introduced into the eukaryotic cells by homologous recombination with a targeting vector comprising said functional DNA sequence flanked by DNA sequences homologous to the Rosa26 locus, or by site specific recombinase mediated recombination with a recombination vector comprising said functional DNA sequence flanked by a pair of first recombinase recognition sites (RRSs);
- step (a) introducing a functional DNA sequence into the Rosa26 locus of starting eukaryotic cells, preferably said functional DNA sequence is introduced into the eukaryotic cells by homologous recombination with a targeting vector comprising said functional DNA sequence flanked by DNA sequences homologous to the Rosa26 locus, or by site specific recombinase mediated recombination with a recombination vector comprising said functional DNA sequence flanked by
- said functional DNA sequence is a gene expression cassette (a) comprising a gene of interest operatively linked to a promoter, or (b) is a DNA sequence which can be converted into such gene expression cassette;
- the eukaryotic cells are mammalian embryonic stem (ES) cells, preferably are non-human mammalian ES cells;
- eukaryotic cells having a modified Rosa26 locus obtainable by the method of (1) and (2) above; (6) a method for preparing a transgenenic multi-cell organism having a modified
- Rosa26 locus which comprises utilizing the method as defined in (1) and (3) above;
- transgenenic multi-cell organism is a non- human mammal and said method comprises modifying an ES cell as defined in (3) above;
- the targeting vector allows insertion of a single copy of a gene expression cassette, thus avoiding modulation of transgene expression by the arrangement of multiple copies.
- the autosomal Rosa26 locus as insertion site, the expression pattern of the inserted transgene in the non-human animal is predictable; random X-inactivation and/or modulation by chromosomal position effects are avoided. This also eliminates the need to generate and analyse multiple transgenic strains for any given transgene.
- the Rosa26 targeting vector for the site-specific integration can be used for multiple gene expression cassettes.
- Figure 1 Targeted insertion of CreER and CAGGS-Cre-ER into the Rosa26 locus.
- a cassette comprising a Cre-ER operationally linked to a CAGGS promoter or a cassette comprising a splice acceptor site (SA) linked to a Cre-ER are inserted into. the Rosa26 locus via homologous recombination.
- SA splice acceptor site
- FIG. 2 Southern Blot analysis of the inducible recombination of the Rosa (reporter).
- A Genomic DNA was isolated from liver (Li) spleen (Sp) and small intestine (Si) of transgenic mice carrying the SA-creER/Rosa-rep . insert or the CAGGS-creER/Rosa-rep insert. To induce the Cre-ER ; recombinase the mice were treated with Tamoxifen
- mice with the SA-creER/Rosa-rep insert were left untreated (untreated). Presence of the reporter band (floxed) and deletion (deleted) of it upon an induced recombination event are indicated.
- B Transgenic mice carrying at one Rosa26 locus a loxP flanked DNA polymerase ⁇ gene segment (por* 70 *) and at the other a SA-creER/Rosa-rep were treated with Tamoxifen (treated). A control group of mice was left untreated (untreated). Genomic DNA from liver (Li), spleen (Sp), kidney (Ki).
- FIG. 3 Western Blot analysis of recombinase and ⁇ -actin expression. Proteins were extracted from rosa(SA-CreER T2 ) and rosa (CAGGS-CreER 1"2 ) mice and analyzed as described in the "Materials and Method" section. The positions of bands representing CreER T2 and actin are indicated.
- FA fat tissue
- Ty Thymus
- Sp spleen
- Br Brain
- Lu lung
- He heart.
- FIG. 4 Fabp-Cre targeting vector.
- An expression cassette, in which the Cre. recombinase is expressed under the control of the Fabpl 4x a "132 promoter is inserted into the Rosa26 targeting vector. This vector was used to insert the Fabp-Cre cassette into the Rosa26 locus by homologous recombination in ES cells.
- Figure 5 ROSA26 locus of the Cre reporter mice carrying a Cre substrate reporter construct. A recombination substrate (Seq ID NO:9) has been inserted in the ROSA26 locus.
- the substrate consists of a CAGGS promoter followed by a cassette consisting of the hygromycin resistance gene driven by a PGK promoter and flanked by loxP recombination sites. This cassette is followed by the coding region for beta- galactosidase, which is only expressed when the hygromycin resistance gene has been deleted by recombination.
- Figure 6 In situ detection of beta-galactosidase in cryosections of different tissues of
- Fabp-Cre/reporter substrate double transgenic mice Mouse tissues were embedded in OCT, frozen and cut into microsections. The sections were stained for beta- galactosidase activity (indicated by the blue color) by X-gal staining, counterstained with Nuclear Fast Red Solution, dehydrated, mounted and photographed. Detailed Descripion of the Invention
- living organisms relates to multi-cell organisms which can be vertebrates such as mammals (e.g. non-human animals such as rodents including mice and rats; and humans) or non-mammals (e.g. fish) or can be invertebrates such as insects or worms, or can be plants (higher plants, algi or fungi). Most preferred living organisms are mice and fish. .
- mammals e.g. non-human animals such as rodents including mice and rats; and humans
- non-mammals e.g. fish
- invertebrates such as insects or worms
- Most preferred living organisms are mice and fish. .
- Eukaryotic cells and “starting eukaryotic cells” according to the present invention include cells isolated (derived) from the above defined living organisms and cultured in vitro. These cells can be transformed (immortalized) or untransformed (directly derived from living organisms; primary cell culture).
- eukaryotic cells also includes mono-cellular eukaryotic cells such as yeasts, etc.
- the eukaryotic cells are derived from , a multi-cell organism including vertebrates, invertebrates and plants, preferably is a vertebrate cell, more preferably is derived from a mammal, including rodents such as mouse, rat, etc., or a fish such as zebrafish.
- the functional DNA sequence comprises a gene encoding a protein/peptide of interest (i.e. is a expressible and translatable DNA sequence), more preferably said functional DNA sequence is a gene expression cassette (a) comprising a gene of interest operatively linked to a promoter, or (b) is a DNA sequence which can be converted into such gene expression cassette (i.e. into an operatively linked "promoter-gene of interest" construct, e.g. by subsequent modification reactions after its integration).
- a gene expression cassette comprising a gene of interest operatively linked to a promoter
- b is a DNA sequence which can be converted into such gene expression cassette (i.e. into an operatively linked "promoter-gene of interest" construct, e.g. by subsequent modification reactions after its integration).
- the gene of interest within the gene expression cassette can be any gene coding for a certain protein/peptide of interest, including, but not limited to, recombinases, reporter genes, receptors, signaling molecules, transcription factors, pharmaceutically active proteins and peptides, drug target candidates, disease causing gene products, toxins, etc.
- the promoter of the gene expression cassette (which is a heterologous promoter relative to the Rosa26 locus) preferably is a ubiquitous or tissue specific promoter, either constitutive or inducible.
- the ubiquitous promoter in the vector according to the invention is preferably selected from polymerases I, II and III dependent promoters, preferably is a polymerase II or III dependent promoter including, but not limited to, a
- CMV promoter CMV promoter
- CAGGS promoter a CAGGS promoter
- a snRNA promoter such as U6
- a RNAse P RNA promoter such as HI
- a tRNA promoter a 7SL RNA promoter
- 5 S rRNA promoter a 5 S rRNA promoter
- Particularly preferred ubiquitous promoters are CAGGS, hCMV, PGK.
- Preferred tissue specific promoters are FABP (Saam & Gordon, J. Biol. Chem., 274:38071-38082
- Col2A (Ovchinnikov et al., Genesis, 26: 145-146 (2000)); preferred inducible promoter systes are Mx (K ⁇ hn et al. Scinence, 269: 1427-1429 (1995)), tet (Urlinger et al., Proc.
- Suitable inducible promoters are the above-mentioned promoters containing an operator sequence including, but not limited to, tet, Gal4, lac, etc.
- the targeting vector, recombination vector, functional DNA sequence or . gene expression cassette may further comprises one ore more additional functional sequences including but not limited to (selectable) marker genes (such as the neomycin phosphotransferase gene of E.
- coli transposon, etc. recombinase recognition sites (which in case of the recombination vector differ from the first recombinase recognition sites and which include loxP, FRT, variants thereof, etc.), poly A signals (such as synthetic polyadenylation sites, or the polyadenylation site of human growth hormones, etc.), splice acceptor sequences (such as a splice acceptor of adenovirus, etc.), introns, tags for protein detection, enhancers, selection markers, etc.
- poly A signals such as synthetic polyadenylation sites, or the polyadenylation site of human growth hormones, etc.
- splice acceptor sequences such as a splice acceptor of adenovirus, etc.
- introns tags for protein detection, enhancers, selection markers, etc.
- methods (1) to (3) of the invention comprise homologous recombination. It is then preferred that the DNA sequences homologous to the Rosa26 locus are 0.2 to 20 kB, preferably 1 to 10 kB long.
- the eukaryotic cells are derived from mouse, the DNA sequences homologous to the Rosa26 locus are derived from the 5' and 3' flanking arm of the mouse Rosa26 locus, preferably said homologous DNA sequences having the sequences shown in SEQ ID NO:4 and 5, respectively, and the promoter is a CAGGS-promoter, most preferably the targeting vector has the sequence shown in SEQ ID NO:7.
- methods (1) to (3) of the invention comprise recombinase mediated recombination.
- the insertion of transgenes or DNA segments into the genome can be mediated by site specific recombination (Fukushige & Sauer, Proc. Natl. Acad. Sci. USA 89(17): 7905-9 (1992)).
- a site specific recombinase like cre or FLP recombines two recognition target sites like loxP or FRT, respectively.
- the use of two incompatible recognition target sites F3 or F5, Schlake & Bode, Biochemistry, 1994 Nov. 1, 33(43): 12746-51) or inverted recognition target sites (Feng et al., J. Mol. Biol.
- recombinase mediated cassette exchange a FLP based RMCE system is inserted into the Rosa26 locus.
- Said recombinase mediated recombination preferably comprises the steps:
- the RRS are loxP or FRT sites or variants thereof (such as single mutant recognition sited lox66 and lox71 (Albert et al., The Plant J. 7:649-659 (1995)); and/or (ii) the acceptor DNA comprises a negatively selectable marker (e.g. herpes simplex virus thymidin kinase gene, etc.) and or (iii) the donor DNA comprises an inactive positive selection marker . (e.g. neomycin phosphotransferase, etc.).
- a negatively selectable marker e.g. herpes simplex virus thymidin kinase gene, etc.
- the donor DNA comprises an inactive positive selection marker .
- an inactive positive selection marker e.g. neomycin phosphotransferase, etc.
- selectable markers it is referred to U.S. Patents Nos. 5,487,932 and 5,464,763 which are hereby incorporated in their entirety.
- the expression cassette in a particularly preferred embodiment of the methods (1) to (3), and in particular if the method comprises homologous recombination, the expression cassette
- (i) is free of a transcriptional stop signal 5' to the (heterologous) promoter of the cassette (i.e. is a non-protected cassette); and/or- (ii) ' the exogenous promoter is a ubiquitous (constitutive or inducible) promoter.
- the methods (1) to (3) may further (besides step (a) defined above) comprise one or more of the steps (b) isolating the eukaryotic cells, preferably the ES cells having the desired fuctional DNA sequence integrated into the Rosa26 locus; and/or (c) modifying the integrated functional DNA sequence and isolating (ES) cells having the desired modified functional DNA sequence.
- the steps (a) and (b) of the methods (1) to (3) are preferably performed in vitro.
- the step (c) may be performed in vitro and in vivo.
- the invention also provides a method for preparing a transgenenic multi-cell organism having a modified Rosa26 locus which comprises utilizing the method as defined in (1) to (3) above.
- the ES cells may subsequently processed according one or more of the following steps:
- transgenic non-human animals carrying one or more functional genes of interest at the Rosa26 locus are generated (viz. by well known breeding procedures).
- transgenic multi-cell organisms and non-human mammals obtainable by the method (6) and (7), respectively; preferably have an operatively functional gene expression cassette (as defined above) integrated into its Rosa26 locus.
- Such transgenic multi-cell organisms and non-human mammals are suitable for gene function studies, drug development, as disease model animals, etc.
- CreER Rosa-targeting vector A 129 SV/EV-BAC library (Incyte Genomics) was screened with a probe against exon2 of the Rosa26 locus (amplified from mouse genomic DNA using Rscreenls (GACAGGACAGTGCTTGTTTAAGG) (SEQ ID NO: l) and Rscreenlas (TGACTACACAATATTGCTCGCAC) (SEQ ID NO:2)). Out of the identified BACclone a 11 kb EcoRV subfragment was inserted into the Malawi site of pBS.
- Two fragments (a 1 kb SacII/Xbal- and a 4 kb Xbal-fragment) were used as homology arms and inserted into a vector containing a FRT-flanked neomycin resistance gene (unpublished) to generate the basic Rosa26 targeting vector.
- the CAGGS-promoter (SEQ ID NO:6, nucleotides 1-1616) or a splice acceptor site (SA) from adenovirus (Friedrich G., Soriano P., Genes Dev., 5:1513-23 (1991)) were inserted between the 5' arm and the FRT flanked neomycin resistance gene.
- the CreER T2 and a polyadenylation site (pA; SEQ ID NO:6, nucleotides 3921-4099) were cloned 3' of the SA or the CAGGS-promoter.
- the vector is free of a transcriptional stop sequence 5' to the CAGGS-promoter
- FABP-Cre Rosa-targeting vector (SEQ ID NO:8): The splice accetpr site from adenovirus (SEQ ID NO:8, nucleotides 18569-18689) was inserted into the basic Rosa26 targeting vector described in 1. above.
- mice All mice were kept in the animal facility at Artemis Pharmaceuticals GmbH in microisolator cages (Tecniplast Sealsave). B6D2F1 Mice for the generation of tetraploid blastocysts were obtained from Janvier. The polb nox /rosa(CreER T2 ) and ect2 nox /rosa(CreER T2 ) mice were generated by breeding of rosa(CreER T2 ) ES mice with 6T14 (Gu et al., Science, 265, 103-106.), respectively.
- mice by tetraploid embryo complementation The production of mice by tetraploid embryo complementation was essentially performed as described (Eggan et al., Proc Natl Acad Sci USA, 98, 6209-6214.).
- Ligand administration 100 mg Tamoxifen -free base (Sigma, T5648) was suspended in 100 ⁇ l Ethanol and solved in 1 ml sunflower oil (Sigma). This 10 mg/100 ⁇ l tamoxifen solution was sonicated for 1-2 minutes and then stored at -20°C. For p.o. administration the solution was thawed at 55°C and administrated to 4-8 week old mice by a feeding needle (FST Fine Science. Tools GmbH, 18061-20).
- Western blot analysis Western blot analysis was performed using SDS-PAGE (NuPAGE, Invitrogen) and the Breeze Immunodetection System (Invitrogen) according to the manufacturer protocols. Immunodetection was done using sc-543 (HC-20, Santa Cruz Biotechnology, Inc.) against ER, PRB-106C against cre, actin sc-1616 Actin (1-19) against actin and rabbit polyclonal IgG (Santa .Cruz Biotechnology, Inc.) antibodies.
- tissue sections To detect beta-galactosidase activity, tissues were embedded in Tissue Tec OCT (Sakura Finetek Europe B.V., The Netherlands), frozen on dry ice and cut into microsections. The sections were mounted onto slides and dried for 1-4 hours at room temperature. Sections were, fixed for 5 min at room temperature in fixing solution (0,2% glutaraldehyde, 5 mM EGTA, 2mM MgCI 2 in 0.1 M PB ((0.1 M K 2 HPO 4 , pH 7.3)) and washed three times for 15 min at room temperature in washing buffer (2 mM MgCI 2 , 0.02% Nonidet-40 in 0.1 M PB).
- fixing solution 0.,2% glutaraldehyde, 5 mM EGTA, 2mM MgCI 2 in 0.1 M PB ((0.1 M K 2 HPO 4 , pH 7.3)
- tissues were stained for beta-galactosidase activity over night at 37°C using X-Gal solution (0.6 mg/mf X-Gal (predissolved in DMSO), 5 mM potassium hexacyanoferrat III, 5 mM potassium hexacyanoferrat II, in washing buffer).
- X-Gal solution 0.6 mg/mf X-Gal (predissolved in DMSO), 5 mM potassium hexacyanoferrat III, 5 mM potassium hexacyanoferrat II, in washing buffer.
- -Sections were washed twice for 5 min at room temperature in PBS, counterstained with Nuclear Fast Red Solution for 10 min, rinsed shortly in aqua dest, dehydrated through a graded ethanol series and mounted in Eukitt (Sigma, Germany).
- a CreER 1"2 gene (Feil et al., (1997) Biochem Biophys Res Commun., 237, 752-757) under the control of the CAGGS-promoter (Okabe, Fabs Letters 407:313-19 (1997)) was inserted into the rosa26 locus by homologous recombination in ES cells by utilizing the CreER Rosa -targeting vector as described above (Fig. 1).
- a splice acceptor sequence (Friedrich and Soziano (1991), Genes Dev., 9, 1513- 1523) was introduced as a control for the endogenous activity of the rosa26 gene promoter (Fig. 1).
- a / ⁇ xP-flanked hygromycin resistance gene was introduced into the second allele of rosa26 to provide test substrate for Cre ER T2 (Seibler et al., Nucl.
- CreER T2 / reporter CreER T2 / reporter
- Rosa(CAGGS-CreER T2 / reporter) mice were fed with daily 5 mg
- a Cre gene under the control of the Fabpl 4x at _132 -promoter (SEQ ID NO:8; Fig. 4) was inserted into the Rosa26 locus by homologous recombination in FI ES cells carrying a Cre reporter substrate in the second Rosa26 allele. LacZ expression from the reporter construct (SEQ ID NO:9; Fig 5) is activated upon Cre-mediated recombination.
- Targeted ES cells were injected into tetraploid blastocysts to generate FABP- Cre/reporter-substrate double transgenic ES mice.
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EP04700694A EP1583837A1 (en) | 2003-01-08 | 2004-01-08 | Targeted transgenesis using the rosa26 locus |
AU2004204167A AU2004204167B2 (en) | 2003-01-08 | 2004-01-08 | Targeted transgenesis using the Rosa26 locus |
US10/541,683 US20060205077A1 (en) | 2003-01-08 | 2004-01-08 | Targeted transgenesis using the rosa26 locus |
CA002513034A CA2513034A1 (en) | 2003-01-08 | 2004-01-08 | Targeted transgenesis using the rosa26 locus |
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EP03000249A EP1439234A1 (en) | 2003-01-08 | 2003-01-08 | Targeted transgenesis using the rosa26 locus |
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US43936703P | 2003-01-10 | 2003-01-10 | |
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JP2010528648A (en) * | 2007-06-06 | 2010-08-26 | セレクティス | Meganuclease variants that cleave DNA target sequences from the mouse ROSA26 locus and uses thereof |
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Also Published As
Publication number | Publication date |
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EP1583837A1 (en) | 2005-10-12 |
CA2513034A1 (en) | 2004-07-29 |
US20060205077A1 (en) | 2006-09-14 |
AU2004204167B2 (en) | 2010-04-08 |
EP1439234A1 (en) | 2004-07-21 |
AU2004204167A1 (en) | 2004-07-29 |
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