Patent Application
20090113561
The present invention provides for a new type of gene trap cassettes, which can induce conditional mutations. The cassettes rely on directional site-specific recombination systems, which can repair and re-induce gene trap mutations when activated in succession. After the gene trap cassettes are inserted into the genome of the target organism, mutations can be activated at a particular time and place in somatic cells. In addition to their conditional features, the gene trap cassettes create multipurpose alleles amenable to a wide range of post-insertional modifications. Such gene trap cassettes can be used to mutationally inactivate all cellular genes. In addition, the invention relates to a cell, preferably a mammalian cell, which contains the above mentioned gene trap cassette. Moreover, the invention relates to the use of said cell for identification and/or isolation of genes and for the creation of transgenic organisms to study gene function at various developmental stages, including the adult, as well as for the creation of animal models of human disease useful for in vivo drug target validation. In conclusion, the present invention provides a process which enables a temporally and/or spatially restricted inactivation of all genes that constitute a living organism.
With the complete sequencing of the human and mouse genomes, attention has shifted towards comprehensive functional annotation of mammalian genes (Austin, C. P. et al., Nat. Genet. 36, 921-4 (2004); Auwerx, J. et al., Nat. Genet. 36, 925-7 (2004)). Among the various approaches for addressing gene function, the most relevant for extrapolation to human genetic disease is mutagenesis in the mouse. Although several model organisms have been used in a variety of mutagenesis approaches, the mouse offers particular advantages because its genome structure and organization is closely related to the human genome. Most importantly, mouse embryonic stem (ES) cells, which grow indefinitely in tissue culture, allow the generation of mice with defined mutations in single genes for functional analysis and studies of human disease.
Several mutagenesis strategies have been deployed in mice, ranging from random chemical (ENU) mutagenesis coupled with phenotype driven screens (Cox., R. D. and Brown, S. D., Curr. Opin. Genet. Dev. 13, 278-83 (2003); Brown, S. D. and Balling, R. Curr. Opin. Genet. Dev. 11, 268-73 (2001)) to sequence-based approaches using ES cell technology, such as gene trapping and gene targeting (Floss, T. and Wurst, W., Methods Mol. Biol. 185, 347-79 (2002); Mansouri, A., Methods Mol. Biol. 175, 397-413 (2001)).
Gene trapping is a high-throughput approach that is used to introduce insertional mutations across the mouse genome. It is performed with gene trap vectors whose principal element is a gene trap cassette consisting of a promoterless reporter gene and/or selectable marker gene flanked by an upstream 3′ splice site (splice acceptor; SA) and a downstream transcriptional termination sequence (polyadenylation sequence; polyA). When inserted into an intron of an expressed gene, the gene trap cassette is transcribed from the endogenous promoter in the form of a fusion transcript in which the exon(s) upstream of the insertion site is (are) spliced in frame to the reporter/selectable marker gene. Since transcription is terminated prematurely at the inserted polyadenylation site, the processed fusion transcript encodes a truncated and non-functional version of the cellular protein and the reporter/selectable marker (Stanford, W. L. et al., Nat. Rev. Genet. 2, 756-68 (2001)). Thus, gene traps simultaneously inactivate and report the expression of the trapped gene at the insertion site, and provide a DNA tag (gene trap sequence tag, GTST) for the rapid identification of the disrupted gene. As gene trap vectors insert randomly across the genome, a large number of mutations can be generated in ES cells within a limited number of experiments. Gene trap approaches have been used successfully in the past by both academic and private organizations to create libraries of ES cell lines harboring mutations in single genes (Wiles, M. V. et al., Nat. Genet. 24, 13-4 (2000); Hansen, J. et al., Proc. Natl. Acad. Sci. USA 100, 9918-22 (2003); Stryke, D. et al., Nucleic Acids Res. 31, 278-81 (2003); Zambrowicz, B. P. et al., Proc. Natl. Acad. Sci. USA 100, 14109-14 (2003)). Collectively, the existing resources cover about 66% of all protein coding genes within the mouse genome (Skarnes, W. C. et al., Nat. Genet. 36, 543-4 (2004)). However, the gene trap vectors which have been used to generate the currently available resources induce only null mutations and mouse mutants generated from these libraries can report only the earliest and non-redundant developmental function of the trapped gene. Therefore, for most of the mutant strains the significance of the trapped gene for human disease remains uncertain, as most human disorders result from late onset gene dysfunction. In addition, between 20%-30% of the genes targeted in ES cells are required for development and cause embryonic lethal phenotypes when transferred to the germline, which precludes their functional analysis in the adult (Hansen, J. et al., Proc. Natl. Acad. Sci. USA 100, 9918-22 (2003); Mitchell, K. J. et al., Nat. Genet. 28, 241-9 (2001)).
To circumvent the limitations posed by germine mutations, conditional gene targeting strategies use site-specific recombination to spatially and temporally restrict the mutation to somatic cells (von Melchner, H. and Stewart, A. F., Handbook of Stem Cells, ed. Lanza, R., Vol. 1, pp 609-622 (2004)). The creation of conditional mouse mutants requires the generation of two mouse strains, i.e. the recombinase recognition strain and the recombinase expressing strain. The recombinase recognition strain is generated by homologous recombination in ES cells whereby the targeted exon(s) is (are) flanked by two recombinase recognition sequences (hereinafter “RRSs”), e.g. loxP or frt. Since the RRSs reside in introns they do not interfere with gene expression. The recombinase expressing strain contains a recombinase transgene (e.g. Cre, Flp) whose expression is either restricted to certain cells and tissues or is inducible by external agents. Crossing of the recombinase recognition strain with the recombinase expressing strain deletes the RRS-flanked exons from the doubly transgenic offspring in a prespecified temporally and/or spatially restricted manner. Thus, the method allows the temporal analysis of gene function in particular cells and tissues of otherwise widely expressed genes. Moreover, it enables the analysis of gene function in the adult organism by circumventing embryonic lethality, which is frequently the consequence of gene inactivation. For pharmaceutical research, aiming to validate the utility of genes and their products as targets for drug development, inducible mutations provide an excellent genetic tool. However, targeted mutagenesis in ES cells requires a detailed knowledge of gene structure and organization in order to physically isolate a gene in a targeting vector. Although the completed sequencing of the mouse genome greatly assists targeted mutagenesis, the generation of mutant mouse strains by this procedure is still time consuming, labor intensive, expensive and relatively inefficient as it can handle only one target at a time.
To address this problem a conditional gene trapping strategy as described in WO 99/50426 has been developed. It utilizes a gene trap cassette capable of producing mutations that can be switched on and off in a spatio-temporal restricted manner. These gene trap cassettes comprise suitably arranged frt or loxP recombinase recognition sites, which—when exposed to Flp or Cre, respectively—lead to removal or inversion of the gene trap cassette and thereby to induction or repair of the mutation. However, recombination reactions mediated by conventional site specific recombinases, such as FLPe and Cre are normally reversible (described in the above documents) between identical recombinase recognition sites (e.g., two loxP or two frt sites). In a conditional gene trap setting, where the recombinase effects a “flipping” of the gene trap cassette as described in the above documents, this would mean that equal amounts of sense and antisense products would be generated, so that one half of the targeted alleles carrying the gene trap would be inactivated whereas the other half would still have a functional configuration. Therefore, it is important to shift the equilibrium of the recombinase reaction towards the gene trap inversion that causes gene inactivation.
The shifting of the equilibrium of the recombinase reaction was achieved with a gene trap system comprising two specific recombinase recognition sites capable of unidirectional inversion if exposed to the corresponding recombinase. Suitable recombination systems are for example the Cre/loxP recombination system with mutant loxP recognition sites (e.g., single mutant recognition sites lox66 and lox71; Albert et al., Plant J., 7, 649-659 (1995)), which—if subjected to Cre recombination—generates a double mutant- and a wildtype-loxP site, each on one side of the inverted DNA. Since the latter combination of loxP sites is less efficiently recognised by the Cre-recombinase, the inversion is predominantly unidirectional.
An only predominantly unidirectional inversion, however, has the disadvantage that a significant number of non-inverted cassettes will also be present. While this is acceptable for cultured cells, it cannot be tolerated in the living animal.
WO 02/088353 and Schnutgen, F. et al., Nat. Biotechnol. 21, 562-5 (2003) disclose a new strategy for directional site specific recombination termed “flip-excision” (“FIEx”). Two sets of incompatible site-specific recombination recognition sites are flanking the DNA fragment to be inverted, in the same order but in reverse orientation.
However, there is still a need for a site specific recombination strategy that is truly directional to enable successive gene trap cassette inversions to first repair and then re-induce a gene trap mutation.
It was found that the FIEx strategy, if applied on a gene trap cassette of the present invention, allows for true unidirectional inversions. In particular, the gene trap cassettes employ two directional site-specific recombination systems, which, when activated in succession, invert the gene trap cassette from its mutagenic orientation on the sense, coding strand to a non-mutagenic orientation on the anti-sense, non-coding strand and back to a mutagenic orientation on the sense, coding strand. These cassettes rely on directional site-specific recombination systems, and can induce conditional mutations in most genes expressed in mouse embryonic stem (ES) cells. They were used to assemble the largest library of ES cell lines with conditional mutations in single genes yet assembled, presently totaling 1,000 unique genes. Moreover, it could be shown that mutations induced by these vectors in ES cells can be both repaired and re-induced by site-specific recombination.
The present invention thus provides:
(1) a gene trap cassette capable of causing conditional mutations in genes, which comprises a functional DNA segment (FS) inserted in a mutagenic or non-mutagenic manner, in sense or antisense direction relative to the gene to be trapped, said FS being flanked by the recombinase recognition sequences (RRSs) of at least two independent directional site-specific recombination systems, wherein each system
(i) comprises two pairs of heterotypic RRSs, said RRSs being oriented in opposite orientation and the RRSs of the two pairs being lined up in opposite order on both sides of the FS, and
(ii) is capable of inverting FS by means of a recombinase mediated flip-excision mechanism;
(2) a preferred embodiment of the gene trap cassette defined in (1) above, which comprises two functional DNA segments,
A: Schematic representation of the retroviral gene trap vectors. LTR, long terminal repeat; frt (yellow triangles) and F3 (green triangles), heterotypic target sequences for the FLPe recombinase; loxP (red triangles) and lox511 (purple triangles), heterotypic target sequences for the Cre recombinase; SA, splice acceptor; βgeo, β-galactosidase/neomycinphosphotransferase fusion gene; pA, bovine growth hormone polyadenylation sequence; TM, human CD2 receptor transmembrane domain; Ceo, human CD2 cell surface receptor/neomycinphosphotransferase fusion gene.
B: Conditional gene inactivation by a SAβgeopA cassette. The SAβgeopA cassette flanked by recombinase target sites (RTs) in a FIEx configuration is illustrated after integration into an intron of an expressed gene. Transcripts (shown as grey arrows) initiated at the endogenous promoter are spliced from the splice donor (SD) of an endogenous exon (here exon 1) to the splice acceptor (SA) of the SAβgeopA cassette. Thereby the βgeo reporter gene is expressed and the endogenous transcript is captured and prematurely terminated at the cassette's polyadenylation sequence (pA) causing a mutation. In step 1, FLPe inverts the SAβgeopA cassette onto the anti-sense, non-coding strand at either frt (shown) or F3 (not shown) RTs and positions frt and F3 sites between direct repeats of F3 and frt RTs, respectively. By simultaneously excising the heterotypic RTs (step 2), the cassette is locked against re-inversion as the remaining frt and F3 RTs cannot recombine. This reactivates normal splicing between the endogenous splice sites, thereby repairing the mutation. Cre mediated inversion in steps 3 and 4 repositions the SAβgeopA cassette back onto the sense, coding strand and reinduces the mutation. Note that the recombination products of steps 1 and 3 are transient and transformed into the stable products of step 2 and 4, respectively (Schnütgen, F. et al., Nat. Biotechnol. 21, 562-5 (2003)).
A and B: ES cells were infected with FlipROSAβgeo virus and selected in G418. X-Gal positive sub-lines (blue) were electroporated with FLPe (A) or Cre (B) expression plasmids and stained with X-Gal after incubating for 10 days. DNA extracted from blue and white sub-lines was subjected to a multiplex PCR to identify inversions. Primer positions within FlipRosaβgeo are indicated by large arrows; allele specific amplification products are visualized on ethidium bromide stained gels to the right. Legend: t, trapped allele; inv, inverted allele; M, molecular weight marker (1 kb+ladder, Invitrogen); lanes 1-3, parental FlipRosaβgeo trapped ES cell line; lanes 4-6, FLPe (A) and Cre (B) inverted sub-line.
C: sub-lines of the FS4B6 ES cell line harboring Cre or FLPe inverted gene trap insertions were electroporated with both FLPe and Cre expression plasmids. The amplification products obtained from the progeny lines by allele specific PCR are visualized on the ethidium bromide stained gel to the right. Legend: t, trapped allele; inv, inverted allele; re-inv, re-inverted allele; M, molecular weight marker (1 kb+ladder, Invitrogen); FS4B6 (lanes 1-3), parental FlipRosaβgeo trapped ES cell line; FS4B6C14 (lanes 4-6), Cre inverted sub-line; FS4B6F14 (lanes 7-9), FLPe inverted sub-line.
A: X-Gal staining (top) and allele specific PCR amplification products (bottom) from the trapped RBBP7 locus in trapped (t), inverted (inv) and re-inverted (re-inv) Q017B06 cell lines. Primers used for the multiplex PCR reactions were identical to those shown in the diagrams of
B: RT-PCR for the amplification of RBBP7 wild-type and trapped fusion transcripts expressed in Q017B06 cells before and after exposure to FLPe and Cre recombinases. The positions of the primers used are shown on top whereby U19=5′-GCT CTT GAC TAG CGA GAG AGA AG-3′ (SEQ ID NO: 12), B32=5′-CAA GGC GAT TAA GTT GGG TAA CG-3′ (SEQ ID NO:13), U34=5′-CCA GAA GGA MG GAT TAT GC-3′ (SEQ ID NO:14), and U35=5′-ACA GAG CM ATG ACC CM GG-3′ (SEQ ID NO:15). Amplification products are visualized below on ethidium bromide stained gels. Amplification of the RNA polymerase II transcript (RNApol II) serves as a positive control. wt, parental ES cells; t, trapped Q017B06 cells; inv, inverted Q017B06 sub-line; re-inv, re-inverted Q017B06 sub-line; endo, endogenous transcript; fus, fusion transcript.
C: Western blot analysis of the RBBP7 protein expressed in Q017B06 cells. Crude cell lysates from the F1 (wt), Q017B06 (t), inverted Q017B06 (inv) and re-inverted Q017B06 (re-inv) ES cells were resolved by SDS-PAGE and analyzed by Western blotting using the anti-RbAp46 antibody. The anti-lamin A antibody served as a loading control.
A: Allele specific PCR of the trapped Glt28d1 locus in trapped (t), inverted (inv) and re-inverted cell lines.
B: RT-PCR of Glt28d1 wild type and Glt28d1/gene trap fusion transcripts expressed in M117B08 cells before and after exposure to Cre and FLPe recombinases. The position of the respective primers within the trapped gene are shown on top whereby M117B8s=5′-GAG AGT GCT GGC CAG CTG GAA C-3′ (SEQ ID NO:16), G01=5′-CAA GTT GAT GTC CTG ACC CM G-3′ (SEQ ID NO:17), and M117B8as1=5′-CCA CCA TAC TCC ACA CAC TCT G-3′ (SEQ ID NO:18). Amplification products are visualized on ethidium bromide stained gels below. Amplification of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcript serves as a positive control. wt, parental F1 ES cells; t, trapped M117B08; inv, inverted M117B08 sub-line; re-inv, re-inverted M117B08 sub-line; endo, endogenous transcript; fus, fusion transcript.
C: Northern blot analysis of Glt28d1 transcripts expressed in M117B08 cells. 2 μg of polyadenylated RNAs from F1 (wt), Q017B06 (t), inverted M117B08 (inv) and re-inverted M117B08 (re-inv) ES cells were fractionated on 1% formaldehyde/agarose gels and hybridized to a 32P-labeled Glt28d1 c-DNA probe. The Glt28d1 probe was obtained by asymmetric RT-PCR using a reverse primer in exon 10 to amplify sequences upstream of the insertion site. The loading of each lane was then assessed by using a GAPDH probe. Legend: see
“Target Gene” defines a specific gene consisting of exons and at least one intron to be trapped by a gene trap vector.
“Gene disruption and selection cassette (GDSC)” refers to genetic elements comprising from 5′ to 3′ a splice acceptor sequence, a reporter and/or selection gene and a polyadenylation sequence.
A “further” or “third” recombinase in the context of present application is a recombinase, which does not interfere with said at least two recombination systems of embodiments (1) and (2).
“Gene trapping” refers to a random mutagenesis approach in functional genomics and is based on the random integration of a gene disruption and selection cassette into a genome.
“Gene targeting” is a gene specific mutagensis approach in functional genomics and is based on the insertion of a GDSC in combination with an independently expressed selection cassette into the genome by homologous recombination (this is for targeting non-expressed genes).
“Targeted trapping” refers to a gene specific mutagenesis approach in functional genomics and is based on the insertion of a GDSC into the genome by homologous recombination (this is for targeting expressed genes).
“Gene trap vector” refers to a promoterless gene trapping construct which inserts a GDSC into an intron, so that it induces a fusion transcript with the targeted endogenous gene.
“Reporter gene” refers to a gene encoding for a gene product (e.g. CAT, βgalactosidase, βgeo, GFP, EGFP, alkaline phosphatase) that can be readily detected by standard biochemical assays.
“Selectable marker gene” refers to a gene, which is transduced into a cell (i.e. transfected or infected) where its expression allows for the isolation of gene trap vector-expressing cells in media containing a selecting agent, e.g. neomycin, puromycin, hygromycin, HSV-thymidine kinase.
“PolyA” (A=adenylic acid) refers to a nucleic acid sequence that comprises the AAUAAA consensus sequence, which enables polyadenylation of a processed transcript. In a gene disruption or selection cassette (GDSC), the polyA sequence is located downstream to the reporter and/or selectable marker gene and signals the end of the transcript to the RNA-polymerase.
“Splicing” refers to the process by which non-coding regions (introns) are removed from primary RNA transcripts to produce mature messenger RNA (mRNA) containing only exons.
“5“splice site” (splice donor, SD)” and “3′ splice site” (splice acceptor, SA) refer to intron flanking consensus sequences that mark the sites of splicing.
“inversion” refers to a case wherein the GDSC segment is excised from the gene and reinserted in an orientation opposite to its original orientation, so that the gene sequence for the segment is reversed with respect to that of the rest of the chromosome. Said inversions can by accomplished by using recombinase enzymes (e.g. Cre, FLPe).
“ROSA” (Reverse-Orientation-Splice-Acceptor) refers to a gene trap cassette inserted into a retroviral backbone in reverse transcriptional orientation relative to the retrovirus.
“homotypic RRSs” refer to site specific recombinase target sequences that are identical and can recombine with one another in presence of the appropriate recombinase (eg. loxP/loxP, lox5171/lox5171, frt/frt, F3/F3).
“heterotypic RRSs” refer to site specific recombinase target sequences with affinity to the same recombinase (e.g. Cre or FLPe) that are not identical (e.g. loxP/lox5171, frt/F3) and cannot recombine with one another in presence of the appropriate recombinase.
An “organism” in the context of the present invention includes eucaryotes and procaryotes. Eucaryotes within the meaning of the invention include animals, human beings and plants. Further, the animal is preferably a vertebrate (such as a mammal or fish) or an invertebrate (such as an insect or worm). In one particularly preferred aspect of the invention, the vertebrate is a non-human mammal, such as a rodent, most preferably is a mouse.
“Cells, cell cultures and tissues” in the context of present invention are derived from an organism as defined above.
The gene trap cassettes (1) and (2) are hereinafter described in more detail. They preferably comprise the structure
5′-L1-A-L2-B-L3-C-L4-FS-L3-D-L4-E-L1-F-L2-3′,
wherein
L1 and L2 are the heterotypic RRSs of the first site-specific recombination system,
L3 and L4 are the heterotypic RRSs of the second site-specific recombination system, both arrayed on either site of the FS in opposite orientations relative to each other and
A to F are independently from each other either a chemical bond or a spacer polynucleotide. Preferably, (i) B and E are chemical bonds; and/or (ii) at least either A or F and either C or D is a spacer polynucleotide. In other words, spacer polynucleotides are not required between L1 and L2 or between L3 and L4 on both sides of the FS, as spacer polynucleotides on one side are sufficient. Furthermore, there are no spacers required between L2 and L3 or L4 and L1.
It is moreover preferred that in the gene trap cassette the RRSs of said at least two independent directional site-specific recombination systems are recognized by recombinases selected from the site specific recombinases Cre or Dre of bacteriophage P1, FLP recombinase of Saccharomyces cerevisiae, R recombinase of Zygosaccharomyces rouxii pSR1, the A recombinase of Kluyveromyces drosophilarium pKD1, the A recombinase of K. waltii pKW1, the integrase λ Int, the recombinase of the GIN recombination system of the Mu phage, the bacterial p recombinase, and variants thereof. Preferably, the two recombinases are Cre and FLPe, or their natural or synthetic variants. Most preferably, at least two recombinases are Cre and FLPe.
Site specific recombinase variants refers to derivatives of the wild-type recombinases and/or their coding sequence which are due to truncations, substitions, deletions and/or additions of amino acids or nucleotides, respectively, their respective sequences. Preferably, said variants are due to homologous substitution of amino acids or degenerated codon usage. The said Cre recombinase of bacteriophage P1 (Abremski et al., J Biol Chem 216, 391-6 (1984)) is commercially available. Concerning the Dre recombinase it is referred to Sauer B, McDermott J., Nucleic Acids Res. 32:6086-6095 (2004).
Furthermore the minimum length of the spacer polynucleotides A to F is 30 nt, preferably 70 nt, most preferably about 86 nt if the two pairs of RRSs are frt/F3 and about 46 nt if the two pairs of RRSs are loxP/lox5171. The spacer nucleotides can be up to several kilobases in length and can be a functional gene or cDNA, such as genes or cDNAs coding for selectable marker and/or reporter proteins.
In a particularly preferred embodiment of the invention one recombinase is Cre recombinase and L1 and L2, or L3 and L4 are selected from LoxP, Lox66, Lox71, Lox511, Lox512, Lox514, Lox5171, Lox2272 and other mutants of LoxP including LoxB, LoxR and LoxL, preferably from LoxP (SEQ ID NO:5), Lox511 (SEQ ID NO: 6), Lox 5171 (SEQ ID NO:7) and Lox2272 (SEQ ID NO:8). More preferably, at least one of L1 and L2, or L3 and L4 is selected from Lox5171 and Lox2272. Most preferably, L1 (or L3) comprises a LoxP sequence as shown in SEQ ID NO:5 and L2 (or L4) comprises a Lox5171 sequence as shown in SEQ ID NO:7, or vice versa, or L1 (or L3) comprises a loxP sequence and L2 (or L4) comprises a lox2272 sequence as shown in SEQ ID NO:8, or vice versa, or L1 (or L3) comprises a Lox5171 and L2 (or L4) comprises a Lox2271 sequence, or vice versa. sequence as shown in SEQ ID NO:7, or vice versa; and/or the other recombinase is FLPe recombinase and L3 and L4, or L1 and L2 are selected from frt, F3 and F5, preferably L3 (or L1) comprises a frt sequence as shown in SEQ ID NO:9 and L4 (or L2) comprises a F3 sequence as shown in SEQ ID NO:10, or vice versa.
In a preferred embodiment of the invention, the functional DNA segment of the construct (1) further comprises one or more of the following functional elements: splice acceptor, splice donor, internal ribosomal entry site (IRES), polyadenylation sequence, a gene coding for a reporter protein, a toxin, a drug resistance gene and a gene coding for a further site specific recombinase. More preferably, the functional DNA segment comprises at least a splice acceptor and a polyadenylation sequence. Suitable splice acceptors include, but are not limited to, the adenovirus type 2 splice acceptor of exon 2 at positions 6018 to 5888 of SEQ ID NOs:1 and 2; suitable donors include, but are not limited to, the adenovirus exon 1 splice donor; suitable IRES include, but are not limited to, that of the ECM virus; and suitable polyadenylation sequences are the polyadenylation sequence of the bovine growth hormone (bpA or bGHpA) such as the sequence of bpA present in positions 1974-1696 of SEQ ID NOs:1-4.
Suitable reporter genes include, but are not limited to, E. coli β-galactosidase, fire fly luciferase, fluorescent proteins (e.g., eGFP) and human placental alkaline phosphatase (PLAP). Suitable resistance genes include, but are not limited to, neomycin phosphotransferase, puromycin and hygromycin resistance genes. In a preferred aspect, a fusion gene between reporter and resistance gene is used, like the βgalactosidase/neomycinphosphotransferase fusion gene (βGeo) in positions 5886-1993 of SEQ ID NO:1 or the human CD2 cell surface receptor/neomycinphosphotransferase fusion gene (Ceo) in positions 3566-1997 of SEQ ID NO:3.
In a further preferred embodiment of the present invention, the construct (1) further comprises a selection DNA segment suitable for selecting for genes having an incorporated gene trap cassette, said selection DNA segment comprising a reporter or resistance gene and flanking recombinase recognition sites in the same orientation. Suitable resistance genes are those mentioned above, provided, however, that they do not interfere with the resistance gene of the functional DNA segment. Suitable recombinase recognition sites in same orientation include, but are not limited to, loxP and mutants thereof (see SEQ ID NOs:5 to 8), frt and mutants thereof (see SEQ ID NOs:9 to 11), provided, however, that these RRSs do not interfere with the RRSs of the functional segment. Thus, suitable further (third) site specific recombinases are all recombinases mentioned above, which do not interfere with the RRSs of the first and second site-specific recombination system present in the gene trap cassette.
The present invention provides a site specific recombination system which combines gene trap mutagenesis with site-specific recombination to develop an approach suitable for the large scale induction of conditional mutations in ES cells. The strategy is based on a recently described site-specific recombination strategy (FIEx) (Schnutgen, F. et al., Nat. Biotechnol. 21, 562-5 (2003)), which enables directional inversions of gene trap cassettes at the insertion sites. By using gene trap integrations into X-chromosomal genes, we have shown that gene trap vectors equipped with the FIEx system cause mutations that can be repaired and re-induced. Thus, ES cell lines expressing these gene trap vectors can be used for generating mice either with null- or conditional mutations. For example, to obtain straight knock-outs the cell lines can be converted directly into mice by blastocyst injection. However, to obtain conditional mutations, one would first repair the mutation in ES cells, preferentially with FLPe to reserve the more efficient Cre for in vivo recombination, and then proceed to mouse production. Resulting mice would lack germline mutations but would be vulnerable to somatic mutations inducible by Cre. Depending on the type of Cre and the form of its delivery the mutations can be re-activated in prespecified tissues at prespecified times.
Due to the inherent recombinase target sites, the vector insertions create multipurpose alleles enabling a large variety of postinsertional modifications by recombinase mediated cassette exchange (RMCE) (Baer, A. and Bode, J., Curr. Opin. Biotechnol. 12, 473-80 (2001)). Examples include replacing the gene trap cassettes with Cre recombinase genes to expand the Cre-zoo, or with point mutated minigenes to study point mutations. A further option is the insertion of toxin genes for cell lineage specific ablations.
The quality of the conditional mutations induced by the gene trap insertions will largely depend on the gene trap's ability to be neutral from its position on the anti-sense, non-coding strand. While in the two examples described here the anti-sense insertions were innocuous, this will presumably not always be the case. Factors likely to influence the anti-sense neutrality include cryptic splice sites and transcriptional termination signals. In line with this, we have shown previously that aberrant splicing induced by an anti-sense gene trap insertion resulted in a partial gene inactivation and an interesting phenotype (Sterner-Kock, A., Genes Dev. 16, 2264-2273 (2002)). Thus, the most likely outcome of anti-sense insertions that interfere with gene expression are hypomorphic mutations, which have a merit of their own. However, in silico analysis failed to identify sequences that might interfere with gene expression from the anti-sense strands of the present vectors, suggesting that the majority of their insertions create bona fide conditional alleles.
By using the vectors in high throughput screens, we have assembled the largest library of ES cell lines with conditional mutations of single protein coding genes, including secretory pathway genes. Presently, it contains about 1,000 potentially conditional alleles (Tab. 1 and 2), which is about ten times the number produced within the last ten years by gene targeting.
Mus musculus nuclear distribution gene C homolog (Aspergillus),
Mus musculus plakophilin 4 (Pkp4), mRNA.
Drosophila)
Mus musculus NRSF/REST gene for neural-restrictive silencer
Mus musculus mRNA similar to thyroid hormone receptor-
Mus musculus mRNA for mKIAA1745 protein.
Mus musculus mRNA for mKIAA1341 protein.
Mus musculus mRNA for mKIAA1020 protein.
Mus musculus mRNA for mKIAA0799 protein.
Mus musculus mRNA for mKIAA0545 protein.
Mus musculus mRNA for mKIAA0433 protein.
Mus musculus mRNA for mKIAA0333 protein.
Mus musculus mRNA for mKIAA0019 protein.
Mus musculus mPcl2 mRNA for polycomblike 2, partial cds.
Mus musculus cDNA clone MGC: 90742 IMAGE: 6827379,
Mus musculus cDNA clone MGC: 61256 IMAGE: 6822178,
Mus musculus cDNA clone MGC: 60532 IMAGE: 30057964,
Mus musculus cDNA clone IMAGE: 5351131, partial cds.
Mus musculus cDNA clone IMAGE: 4036366, partial cds.
Homo sapiens cDNA clone IMAGE: 4813640, partial cds.
H. sapiens hnRNP A1 gene promoter region.
Mus musculus immunoglobulin germline IgK chain
Mus musculus RIKEN cDNA 2310007F21 gene, mRNA (cDNA
Mus musculus RIKEN cDNA 2410002F23 gene, mRNA (cDNA
Mus musculus RIKEN cDNA 2810026P18 gene, mRNA (cDNA
Rattus norvegicus mRNA for GABA-A receptor interacting
Mus musculus Rho guanine nucleotide exchange factor (GEF)
Mus musculus strain C57BL/10 HLA-B associated transcript 1
Homo sapiens chromosome 14 open reading frame 43, mRNA
Mus musculus platelet endothelial tetraspan antigen-3 (Peta3)
Mus musculus chromosome condensation 1, mRNA (cDNA
Mus musculus partial mRNA for CLIP-associating protein
Mus musculus cytoplasmic FMR1 interacting protein 1, mRNA
Mus musculus epithelial protein lost in neoplasm-b (Eplin)
Mus musculus erythrocyte protein band 4.1-like 2, mRNA (cDNA
Mus musculus, frizzled homolog 7 (Drosophila), clone
Mus musculus Ras-GTPase-activating protein SH3-domain
Mus musculus glutathione S-transferase, alpha 4, mRNA (cDNA
Mus musculus hypermethylated in cancer 2, mRNA (cDNA
Homo sapiens OZF gene exon 1.
Rattus norvegicus cDNA clone MGC: 72278 IMAGE: 5598632,
Mus musculus, clone IMAGE: 5324476, mRNA.
Mus musculus oxoglutarate dehydrogenase (lipoamide), mRNA
Mus musculus gene for polyA polymerase, exon 1.
Mus musculus plexin B2, mRNA (cDNA clone MGC: 37720
Mus musculus protein kinase, lysine deficient 1, mRNA (cDNA
Rattus norvegicus NAP-22 mRNA for acidic membrane protein
Mus musculus RNA binding motif protein, X chromosome
Mus musculus RNA transcript from U17 small nucleolar RNA
Mus musculus equilibrative nucleoside transporter 1 gene,
Mus musculus transcription factor E3 (Tcfe3) mRNA, partial
Mus musculus RIKEN cDNA 9530058O11 gene, mRNA (cDNA
Mus musculus vasodilator-stimulated phosphoprotein, mRNA
Mus musculus WD repeat domain 33, mRNA (cDNA clone
Mus musculus DNA segment, Chr 1, University of California at
Mus musculus RIKEN cDNA 4933425L19 gene, mRNA (cDNA
Mus musculus RIKEN cDNA 1810014B01 gene, mRNA (cDNA
Mus musculus RIKEN cDNA 1500015A07 gene, mRNA (cDNA
Mus musculus cDNA clone IMAGE: 6810589, partial cds.
Considering that these gene trap lines were isolated in less than a year, conditional gene trapping seems significantly more efficient than conditional gene targeting. However, analysis of the existing gene trap resources indicates that gene trapping is more efficient than gene targeting only up to about 50% of all mouse genes, after which the mutation rate falls to a level comparable to gene targeting (Skarnes, W. C. et al., Nat. Genet. 36, 543-4 (2004)). Moreover, effective gene trapping is restricted to the approximately 70% of the genes expressed in ES cells (Ramalho-Santos, M. et al., Science 298, 597-600 (2002); Ivanova, N. B. et al., Science 298, 601-4 (2002)). We believe that for a comprehensive mutagenesis of the mouse genome, a balance between gene trapping and gene targeting, performed with generic gene trap cassettes inserted into the targeting vectors, is likely to be the most efficient and cost-effective.
The principal elements of a conditional gene trap cassette of embodiments (1) and (2) of the invention that selects for integrations into expressed genes are (i) a conditional gene disruption segment, containing a 3′ splice site (splice acceptor; SA) and a polyadenylation sequence (polyA) flanked by the RRSs of the two recombination systems, and (ii) a selection segment containing a reporter or selectable marker gene flanked by an upstream SA- and a downstream polyA-site. The selection segment is flanked by two RRSs in same orientation, which are recognized by a further recombinase and is in opposite orientation to the gene disruption cassette. Selection for gene expression with the gene trap cassette of embodiment (1) and (2) yields recombinants in which the reporter gene is fused to the regulatory elements of an endogenous gene. Transcripts generated by these fusions encode a truncated cellular protein which has lost its normal function. Since selection for a gene trap event relies on the expression of the selection cassette, which is by itself mutagenic, it needs to be inverted to the antisense, noncoding strand in embodiment (1) or removed to recreate gene function in embodiment (2). This is achieved in (1) by expressing the first recombinase in recombinants selected for gene trap integrations. In a favoured operational process the conditional gene trap is transduced into ES cells. After selecting for integrations into the introns of expressed genes, the first recombinase is transiently expressed in individual clones to invert the gene trap cassette to the antisense, non-coding strand and thus restore gene function. The resulting clones containing the gene disruption cassette on the antisense, non-coding strand are used to create transgenic mouse strains. Such mouse strains are crossed to mouse strains expressing the second recombinase to obtain doubly transgenic offspring where the gene disruption cassette is re-inverted to its original mutagenic (sense) orientation on the coding strand. Alternatively, the removal of the selection cassette of embodiment (2) is achieved by the first recombinase as follows: the gene trap cassette is transduced into ES cells. After selecting for integrations into the introns of expressed genes, the first recombinase is transiently expressed in individual clones to delete the selection cassette and thus restore gene function. The resulting clones containing only the gene disruption cassette on the antisense, non-coding strand are used to create transgenic mouse strains. Such strains are crossed to mouse strains expressing the second recombinase to obtain doubly transgenic offspring, where the gene disruption cassette is inverted to its mutagenic (sense) orientation on the coding strand.
As a further preferred embodiment the invention provides a conditional gene trap vector that selects for integrations into genes regardless of their expression. In other words, selection for integrations into all genes, expressed and non-expressed, are possible. This is achieved by adding to the original gene disruption cassette a second cassette in which a selection gene is fused to an upstream constitutive promoter and to a downstream 5′ splice site (splice donor) (Zambrowicz et al., Nature, 392, 608 (1998)). Expression of this gene trap is dependent on the acquisition of an endogenous polyadenylation sequence, which occurs by splicing of the selection cassette to the downstream exons of the target gene. Since the process is driven by a constitutive promoter, selection for gene trap integrations is independent of the target gene expression. As with the other conditional gene trap, a favoured operational process is its transduction into ES cells and the generation of mutant mouse strains.
The introduction of the gene trap cassette in the processes of embodiments (4) and (5) of the invention into a suitable cells can be effected by conventional methods including electroporation or retroviral infection. “Suitable cells” refers to appropriate starting cells, including cells pretreated for the introduction.
In a preferred embodiment of the processes (4) and (5), the introduction of the gene trap cassette into the cell is done by homologous recombination. The gene trap cassette used in this embodiment is flanked by homology regions apt for homologous recombination, preferably by homology regions corresponding to a first intron of a target gene. This gene trap cassette modification is also a preferred aspect of embodiments (1) and (2). The cassette is introduced into the ES cell by homologous recombination. Thus, the cassette can be used to introduce conditional mutations into specific target genes.
In a preferred embodiment of the process (5) of the invention said process further comprises one or more of the following steps
In a further preferred embodiment, inversion of the functional DNA segment into a neutral position and the induction of a mutation in the trapped gene by inversion of the functional DNA segment according to steps (iv) and (iv) above is effected by using recombinases for one of said directional site-specific recombination systems of the gene trap cassette. The process (5) is suitable for temporally and/or spatially restricted inactivation of all genes that constitute a living organism and for preparing transgenic non-human mammals, especially transgenic mice. In such process, the gene trap cassette as defined above is introduced into an ES cell. ES-cell derived chimeras may be established by routine measures well known in the art, e. by injecting C57BI/6 blastocysts, breeding the resulting male chimeras to C57BI/6 females, and testing agouti offspring for transgene transmission by tail blotting.
The above process possesses the following advantages over current technology:
The present invention is further illustrated by the following Examples which are, however, not to be construed as to limit the invention.
Plasmids: pFlipROSAβgeo (SEQ ID NO:1) was assembled in pBabeSrf, a modified pBabepuro retroviral vector lacking the promoter and enhancer elements from the 3′LTR (Gebauer, M. et al., Genome Res 11, 1871-7 (2001)). Pairs of the heterotypic frt/F3 and lox511/loxP recombinase target sequences (RTs) were cloned in the illustrated orientation (
The pCAGGS-FLPe expression plasmid was a gift from A. Francis Stewart (Rodriguez, C. I. et al., Nat Genet. 25, 139, (2000)). The pCAGGS-Cre expression plasmid was derived from and pCAGGS-FLPe by replacing the FLPe cDNA with the Cre cDNA of pSG5Cre (Feil, R. et al., Biochem Biophys Res Commun 237, 752 (1997)).
The expression plasmids prFlipRosabgeo and prFlipRosaCeo (SEQ ID Nos:2 and 4, respectively) are based on the plasmids pFlipROSAβgeo and pFlipRosaCeo, respectively, wherein the lox511 sites have been replaced by lox5171 sites. In the following Examples plasmids with lox511 sites are utilized.
ES-cell cultures, infections and electroporations: The [C57BL/6J×129S6/SvEvTac] F1 ES cell lines were grown on irradiated or Mitomycin C treated MEF feeder layers in the presence of 1000 U/ml of leukemia inhibitory factor (LIF) (Esgro®, Chemicon Intl., Hofheim, Germany) as previously described (Hansen, J. et al., Proc Natl Acad Sci USA 100, 9918 (2003)).
Gene trap retrovirus was produced in Phoenix-Eco helper cells by using the transient transfection strategy described previously (Nolan, G. P. & Shatzman, A. R. Curr Opin Biotechnol 9, 447 (1998)). ES cells were infected with the virus containing supernatants at an M.O.I.<0.5 as previously described (Hansen, J. et al., Proc Natl Acad Sci USA 100, 9918 (2003)). Gene trap expressing ES-cell lines were selected in 130 μg/ml G418 (Invitrogen), manually picked, expanded, and stored frozen in liquid nitrogen.
Electroporations were carried out using 1×107 ES cells, 10 μg of plasmid DNA and a 400 μF capacitator (BioRad, Hercules, USA) as previously described (Floss, T. & Wurst, W., Methods Mol Biol 185, 347 (2002)). After incubating for 2 days in medium supplemented with 0.6 μg/ml puromycin (Sigma-Aldrich, Munich, Germany), the cells were trypsinized and seeded at low density (1000 cells/dish) onto 60 mm Petri dishes. Emerging clones were manually picked after 9 days and expanded. The resulting cell lines were used for X-Gal stainings and molecular analyses.
Nucleic acids and protein analyses: PCRs were performed according to standard protocols using 300-500 ng of genomic DNA or 1 μg of reverse transcribed total RNA in a total volume of 50 μl. The primer sequences used are available upon request.
For Northern blotting, polyA+ RNA was purified from total RNA using the Oligotex mRNA-mini-kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. The mRNA (1-2 μg) was fractionated on 1% formaldehyde-agarose gels, blotted onto Hybond N+ (Amersham, Freiburg, Germany) nylon membranes, and hybridized to 32P-labeled cDNA probes (Hartmann Analytic, Braunschweig, Germany) in ULTRAhyb hybridization solution (Ambion, Austin, Tex., USA) according to manufacturer's instructions. The Glt28d1-cDNA probe was obtained by asymmetric RT-PCR (Buess, M. et al., Nucleic Acids Res 25, 2233 (1997)). using an anti-sense primer complementary to exon 10 of the Glt28d1 gene.
Semiautomated 5′RACE and sequencing was performed as previously described (Hansen, J. et al., Proc Natl Acad Sci USA 100, 9918 (2003)). The sequences of the generic and vector-specific primers used are available upon request.
Western blots were performed as previously described (Sterner-Kock, A. et al., Genes Dev 16, 2264-, (2002)), using anti-RbAp46, (Abcam, Cambridge, UK) and lamin A (Santa Cruz, Heidelberg, Germany) primary antibodies.
GTST analysis: GTSTs were analyzed as previously described (Hansen, J., et al., Proc Natl Acad Sci USA 100, 9918, (2003) using the following databases: GenBank (rel. 144), UniGene (build 141), RefSeq (rel. 8) (all at http://www.ncbi.nlm.nih.gov), ENSEMBL v26.33 (http://www.ensembl.org), MGI (http://www.informatics.jax.org/) and GeneOntology (December 2004 release) (http://www.geneontology.org).
Two gene trap vectors were designed for large scale conditional mutagenesis in ES cells. The first vector FlipRosaβgeo contains a classic splice acceptor (SA)-β-galactosidase/neomycintransferase fusion gene (βgeo)-polyadenylation sequence (pA) cassette inserted into the backbone of a promoter- and enhancerless Moloney murine leukemia virus in inverse transcriptional orientation relative to the virus (
A modified version of a recently published site-specific recombination strategy termed FIEx (flip-excision) (Schnutgen, F. et al., Nat Biotechnol 21, 562-5 (2003)) was applied. FIEx uses pairs of inversely oriented heterotypic recombinase target sequences (RTs) such as loxP and lox511 or frt and F3. When inserted upstream and downstream of a gene trap cassette, Cre or FLPe recombinases invert the cassette and place a homotypic RT pair near to each other in a direct orientation. Recombination between this pair of directly repeated RTs excises one of the other heterotypic RTs, thereby locking the recombination product against re-inversion to the original orientation. Thus, by flanking the gene trap cassettes of FlipRosaβgeo and FlipRosaCeo with pairs of heterotypic 10× and frt sites (
To test for recombinase-mediated inversions, several FlipRosaβgeo-trapped ES cell lines were selected for high levels of βgeo expression using X-Gal staining. X-Gal positive (blue) cell lines were then transiently transfected with FLPe or Cre expression plasmids and emerging subclones were stained with X-Gal. As shown in
To test whether the FLPe or Cre inverted cell lines would re-invert following a second recombinase exposure, we re-expressed FLPe and Cre in each of the cell lines and checked their progeny for re-inversions by the allele specific PCR.
To test whether the mutations induced by the conditional gene trap vectors are reversible, we selected the Q017B06 and M117B08 gene trap lines for further analysis. In Q017B06, the FlipRosaβgeo gene trap vector disrupted the retinoblastoma binding protein 7 (RBBP7) gene at the level of the first intron. In M117B08, the FlipRosaCeo gene trap vector disrupted the glycosyltransferase 28 domain containing 1 gene (Glt28d1) in the 10th intron. Both genes are located on the X-chromosome of a male derived ES cell line, which provided a haploid background for the mutational analysis. As shown in
A critical issue that could be addressed with these trapped ES cell lines was whether endogenous gene expression would resume after Cre or FLPe induced inversions. Towards this end, we expressed Cre or FLPe in the Q017B06 and M117B08 cell lines, isolated several sub-lines, and genotyped them by allele-specific PCR (FIG. 3,4 panels A). Inverted sub-lines were then analyzed for RBBP7, Glt28d1 and gene trap cassette expression using RT-PCR in combination with Northern- and Western blotting.
We isolated 4,525 ES cell lines with conditional gene trap insertions and recovered 4,138 gene trap sequence tags by 5′RACE. Of these, 3,257 were derived from FlipRosaβgeo and 881 from FlipRosaCeo integrations. Ninety percent of the FlipRosaβgeo and 99% of the FlipRosaCeo GTSTs belonged to RefSeq annotated genes (Table 1). The number of annotated genes was nearly double that found in our previous analysis (Hansen, J. et al., PNAS 100, 9918 (2003)), reflecting the swift progress in genome annotation. The overall efficiency of trapping was similar to that observed in previous studies, as was the number of preferred insertions sites (i.e., hot spots) (Table 1). Insertions occurred in all chromosomes, including one on the Y chromosome and their number correlated with the number of genes per chromosome (data not shown). Collectively, these observations indicate that the heterotypic 10× and frt sites built into the gene trap vectors do not affect the efficiency of trapping. Regardless of the vector, the vast majority of gene trap insertions occurred into first and second introns, confirming the reported preference of retroviral integrations near the 5′ ends of genes (
This example describes the use of trapped ES cell lines for making mutant mice. ES-cell derived chimeras were generated by injecting C57BI/6 blastocysts with ES cells harboring conditional mutations in the following genes (Table 2): translocase of inner mitochondrial membrane 9 homolog (clone ID: P015F03; acc.# NM—013896), frizzled homolog 7 (clone ID: P016E04; acc# BC049781), strawberry notch homolog 1 (clone ID: P023A01; acc# XM—355637), nucleoporin 214 (clone ID: P023F01; acc# XM—358340), Parkinson disease 7 (clone ID: Q001D04; acc# NM—020569 and YME1-like 1 (clone ID: Q016D06; acc# NM—013771). Male chimeras were obtained with each clone and were bred to C57BI/6 females. Litters were analyzed for germline transmission using the agouti coat color marker and Southern blotting of tail DNA. So far, the clones P015F03 and P016F03 transmitted the mutation to the F1 generation. F1 mice were crossed to a FLPe recombinase expressing strain to neutralize the mutation by inverting the FlipRosabgeo GDSC onto the antisense, non-coding strand. The F2 offspring of these mice are conditional “ready” and can be used to induce tissue specific mutations at prespecified times. This is accomplished by crossing the F2 mice to mice expressing an inducible Cre recombinase under the control of a tissue specific promoter.
| Number | Date | Country | Kind |
|---|---|---|---|
| 04028194.1 | Nov 2004 | EP | regional |
| 05103092.2 | Apr 2005 | EP | regional |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP2005/056282 | 11/28/2005 | WO | 00 | 8/3/2007 |
