Gene traps provide a general strategy to identify genes exhibiting discrete patterns of expression during development and differentiation. The basic design of gene trap vectors has been based on the introduction of a promoterless reporter gene, e.g., xcex2-galactosidase, into embryonic stem (ES) cells, which could only be expressed if the reporter gene has integrated in the right frame and orientation within a transcriptional unit. To improve the chances of expression, the reporter gene has been placed. downstream of a splice acceptor (SA) sequence allowing expression to occur when the reporter gene is integrated within an intron. Integration results in reporter gene expression that reflects the expression pattern of the endogenous gene or is influenced by nearby transcriptional regulatory elements.
One method of constructing trap vectors has been to use as a background a retroviral vector. Retroviruses integrate into the genome with no rearrangements of flanking sequences. This is not always the case when DNA is introduced by microinjection and perhaps other methods. An additional advantage of using a retroviral vector has been that the sites of proviral integration are often found close to hypersensitive sites (Vijaya et al., J. Virol. 60:683-692 (1986); Rohdewohld et al., J. Virol. 61:336-343 (1987)).
In one particular design the reporter gene is inserted in Reverse Orientation (with respect to retroviral transcription) downstream of a Slice Acceptor sequence, and the resultant mice are referred to as ROSA. Using this gene trap design several different reporter genes have been used in embryonic stem cells as a genetic screen to identify and mutate developmental genes in mice (Friedrich and Soriano, Genes and Development 5:1513-1523 (1991)). Using the ROSA gene trap design many gene trap transgenic lines have been derived that display various expression patterns in embryos at different developmental stages. Among the transgenic lines obtained have been certain promoters which appear to be ubiquitously expressed.
Gene targeting in murine embryonic stem cells has allowed the production of mice with specific gene deletions. This technique has been used in studies to try and determine the functional identification of gene products. Conventional gene knockout techniques have provided mice that inherit genetic deletions in all cell types in a regionally and temporally unrestricted manner which can lead to severe developmental defects and premature death of the knock-out animals. Several model systems have been developed which attempt to take advantage of the cell-type or tissue-type restricted expression of certain promoters operatively associated with a recombinase gene in combination with a gene of interest that has been flanked by recombinase recognition sequences. In this system the recombinase is expressed under the control of the cell-type or tissue-type specific promoter and when expressed results in the excision of the gene of interest. Because the temporal nature of cell-type and/or tissue-type specificity of the promoters is not known with any certainty results obtained with this system are suspect. Further, as these promoters are not active in all cells or in all tissues of an animal they are not-as useful for examining conditional mutations in genes.
The present invention provides methods and vector constructs which quite unexpectedly provide means to position a gene of interest under the control of an ubiquitously expressed promoter and a means to confirm the nature of the expression of the promoter sequence such that the study of these mutations in transgenic non-human organisms can be accomplished.
The present invention provides methods and vector constructs for the production of genetically engineered non-human animals which ubiquitously express a heterologous DNA segment. In one embodiment of the invention, the methods comprise transforming a pluripotent cell with a DNA construct comprising a heterologous DNA segment in which at least 100 base pairs is homologous with a DNA sequence of an ubiquitously expressed endogenous gene locus of the pluripotent cell, wherein the DNA construct becomes integrated into the gene locus by homologous recombination. Insertion of the DNA construct into the ubiquitously expressed gene locus places the DNA construct under the control of the promoter of the ubiquitously expressed gene locus and does not result in a lethal mutation.
Pluripotent cells which carry the heterologous gene inserted into and under the control of the ubiquitously expressed gene locus promoter are selected and introduced into a developing embryo, at, for example, the blastocyst or morula stage. The embryos are allowed to develop to term and offspring are selected which carry the heterologous DNA segment integrated into the ubiquitously expressed endogenous gene locus and under its promoter.
In a particularly preferred embodiment of the invention, the pluripotent cells are murine embryonic stem cells, zygotes or sperm cells, and the like. Also, in an alternative embodiment, a splice acceptor sequence is operatively associated with the heterologous DNA segment.
The methods of the present invention can be used to produce general deletor and general reporter (alternatively designated a Universal Conditional Reporter (UCR)), animal strains. In one representative embodiment of a general deletor animal, the heterologous DNA segment is a general deletor cassette comprising a gene encoding a recombinase. Optionally, a splice acceptor sequence can be associated with gene encoding the recombinase. The animal produced using the methods of the present invention ubiquitously express the recombinase in essentially all cells and all tissues throughout development. When these mice are crossed with an animal strain which has a gene of interest flanked by recombinase recognition sequences recognized by the recombinase expressed by the general deletor mouse, the gene of interest is excised from the chromosome. It is necessary for the two recombinase recognition sequences to be in the same orientation within a ubiquitously expressed endogenous gene locus. The heterologous DNA segment is positioned within the ubiquitously expressed endogenous promoter such that the reporter cassette expression is under the control of the promoter. A particularly preferred reporter is xcex2-galactosidase.
Representative ubiquitously expressed endogenous gene loci which. can be used in the methods of the present invention include ROSA26, ROSA5, ROSA23, ROSA11, and G3BP (BT5). Other loci can be determined by gene traps or other well known methods.
In an alternative embodiment of the present invention, the general deletor cassette further comprises a positive selection cassette downstream of the heterologous DNA segment. This cassette can be used in the identification of pluripotent cells which have been integrated under control of a ubiquitous promoter. Representative positive selectable markers include neo, gpt and others.
In an alternative embodiment of the general reporter cassette, the DNA stuffer sequence can comprise a promoter operatively associated with a selectable marker. The promoter can be inducible if desired. One particularly preferred promoter is PGK. Particularly preferred recombinase recognition sequences are lox and frt which are recognized by Cre and Flp recombinase, respectively.
The present invention also provides a general targeting vector cassette which comprises at least 100 base pairs of a DNA sequence homologous with an ubiquitously expressed endogenous gene locus and optionally, a negative selection cassette.
Representative gene loci which are ubiquitously expressed and can be used in the present invention include ROSA26, ROSA5, ROSA23, ROSA11 and G3BP (BTS). A particularly preferred negative selection marker is Diphtheria toxin. In a particularly preferred embodiment, 5 kb of the ROSA 26 locus is used to create a general targeting vector cassette.
In another embodiment of the present invention, a general deletor cassette is provided. The general deletor cassette comprises at least 100 base pairs of a DNA sequence homologous with an ubiquitously expressed endogenous gene locus and, optionally, a negative selection cassette. Positioned within the homologous DNA sequence is a gene encoding a recombinase. Preferred recombinase for use in the present invention are Cre and Flp. The recombinase can further be associated with an Internal Ribosome Entry Site (IRES) upstream of the recombinase gene. A particularly preferred IRES is derived from the Encephalomyocarditis virus.
In an alternative embodiment, a splice acceptor sequence is operatively associated with the recombinase gene. Downstream of the recombinase gene, the general deletor vector cassette can further include a positive selection cassette. In a preferred embodiment, the positive selection cassette comprises a promoter, such as PGK, and a positive selectable marker, such as neo or Herpes simplex virus tk. Particularly preferred ubiquitously expressed endogenous gene loci for use in the general deletor cassette are ROSA26, ROSA5, ROSA23, ROSA11, and G3BP (BT5).
In yet another embodiment of the present invention, a conditional deletor cassette is provided. The conditional deletor cassette comprises at least 100 base pairs of a DNA sequence homologous with a conditionally expressed endogenous gene locus, and optionally, a positive or negative selection cassette. Positioned within the homologous DNA sequence is a gene encoding a recombinase as described above. The recombinase can be further associated with an IRES upstream of the recombinase gene. A particularly preferred IRES is derived from the Encephalomyocarditis virus.
In an alternative embodiment, a splice acceptor sequence is operatively associated with the recombinase gene. Downstream of the recombinase gene, the general deletor vector cassette can further include a positive selection cassette. In a preferred embodiment, the positive selection cassette comprises a promoter, such as PGK, and a positive selectable marker, such as neo or Herpes simplex virus tk. A particularly preferred ubiquitously expressed endogenous gene loci for use in the conditional deletor cassette is EphA2.
In still another embodiment of the present invention, a general reporter vector cassette is provided. This cassette comprises at least 100 base pairs of a DNA sequence homologous with an ubiquitously expressed endogenous gene locus and, optionally, a negative selection cassette. Within the DNA sequence is inserted a DNA stuffer sequence flanked by two recombinase recognition sequences in the same orientation positioned upstream of a gene encoding a reporter. In one alternative embodiment of the present invention an IRES is positioned upstream of the gene encoding a reporter. In another alternative embodiment, a splice acceptor is operatively associated with the DNA stuffer sequence. The negative selection cassette is preferred to comprise a promoter operatively associated with a negatively selectable marker. A particularly preferred selectable marker is the Diphtheria toxin gene.
Preferred recombinase recognition sequences include, but are not limited to lox and Frt which are recognition sequences for the recombinases Cre and Flp, respectively.