Animal models for loss and gain of Hox11 function

#This invention is generally in the area of homeobox gene function. In particular, animals deficient in orphan homeobox gene function serve as models for organogenesis and are useful in methods for screening and assessing therapies and compounds in treating orphan homeobox gene deficiency.

#Various genes have been identified that are involved in the ontogenic process, from polarity generation in the fertilized egg (Manseau, L. J. et al., Trends Genet., 5: 400-405 (1989)) to embryonic axis formation (Niehrs, C. et al., Curr. Opin. Genet. Dev., 2: 550-555 (1990)) to gastrulation (Stern, C. D., Curr. Opin. Genet. Dev., 2: 556-561 (1990)) and spatial pattern formation (McGinnis, W. et al., Cell, 68: 283-302 (1992)). A combinatorial gene program appears to pattern the evolutionarily ancient and critical elements of the central nervous system (CNS) and its axial support structures. However, the genetic events that control formation and patterning of individual organs remain principally unknown. Each gene participating in organogenesis could play a role in the development of numerous tissues. Of interest, WT-1 deficient mice fail to develop kidneys but have other abnormalities as well (Kreidberg, J. A. et al., Cell, 74: 679-691 (1993)). Alternatively, following regional specification by a gene network, individual genes might be activated which then direct development of a single organ.

#The phenomenon of "homeosis" is the aberrant development of one body part or segment of an organism into the likeness of another body part. The best studied examples of homeosis occur in the fruit fly (Drosophila). Genetic analysis revealed that mutation in any of a few dozen genes in the fruit fly can lead to homeotic changes, such as the head segment growing legs where antennae belong (Wakinoto et al., Dev. Biol., 81: 51-64 (1981)), or a second thoracic segment developing in place of an abdominal segment (Sanchez-Herrero et al., Nature, 313: 108-113 (1985)). Characterization of these genes revealed that they are involved in controlling segmental identity and that they shared a highly conserved sixty amino acid sequence, now called the homeobox or homeodomain (Gehring, Science, 236: 1245-1252 (1987)).

#FIG. 1a shows schematically a restriction endonuclease map of a phage clone containing the mouse genomic Hox11 gene. FIG. 1b shows schematically a restriction endonuclease map of a 7 kilobase (kb) subclone of the phage clone containing the Hox11 gene and the relative position of Hox11 exons 1, 2 and 3. Open boxes indicate exons. Incomplete box indicates that exon 1 contained the 5' untranslated region of Hox11 which was not sequenced. Restriction sites: B, BglII; Ba, BamHI; Bs, BssHI; E, EcoRI; Ev, EcoRV; H, HindIII; K, KpnI; M, MscI; N, NarI; Nc, NcoI; S, SalI; Sh, SphI; Sp, SpeI; X, XbaI. The SalI sites are from the phage arms and are not found in the genomic DNA.

#FIG. 2 shows a comparison of the amino acid sequences of murine (Sequence ID No. 1) and human (Sequence ID No. 2) Hox11 proteins as deduced from nucleic acid sequence data.

#FIG. 3 shows schematically the targeting construct used to inactivate ("knock out") a genomic mouse Hox11 gene by homologous recombination (indicated by the two sets of crossed lines) and the relative position of external and internal probes used to confirm insertion of targeting construct sequences. PGK-Neo, a neomycin resistance gene driven by a PGK promoter, inserted into a copy of exon 1 of Hox11; HSV-TK, a herpes simplex virus thymidine kinase gene inserted at the 3' end of the coding sequence of exon 3 of Hox11; B, BamHI; E, EcoRI; H, HindIII; N, NarI; S, SalI.

#An effective animal model for deficiency in a gene that controls organogenesis must possess both alleles stably inactivated so that throughout embryogenesis one or more tissues cannot revert to a functional wild-type allele.

#One method of generating animals with an altered genotype is gene targeting, as described by Mansour et al. (Nature, 336: 348-352 (1988)), in which homologous recombination of newly introduced DNA sequence (i.e., the targeting sequence or construct) and a specific targeted DNA sequence residing in the chromosome results in the insertion of a portion of the newly introduced DNA sequence into the targeted chromosomal DNA sequence. This method is capable of generating animals of any desired genotype, and is especially useful for disrupting (i.e., to "knock out") a specific chromosomal gene sequence by inserting a selectable marker into the gene. To knock out a genomic gene, a cloned fragment must be available and intron-exon boundaries within the fragment defined (Mansour et al., 1988). Typically, the targeting construct contains a selectable marker such as Neo (neomycin resistance, see Mansour et al., 1988) flanked by sequences homologous to the chromosomal target DNA, and beyond one of these flanking sequences the herpes simplex virus thymidine kinase gene (HSV-TK, McKnight et al., Nucleic Acids Res., 8: 5949-5965 (1980)). The targeting construct is introduced, e.g., by electroporation, into embryo-derived stem (ES) cells where homologous recombination results in an insertion of the Neo marker, but not the HSV-TK gene, into the targeted chromosomal DNA sequence. The altered ES cells are neomycin resistant and HSV-TK.sup.- and so are able to grow in the presence of both G418 and gancyclovir antibiotics, whereas random insertions contain the HSV-TK gene and are sensitive to gancyclovir (Mansour et al., 1988). Positive ES clones can then be microinjected into blastocysts to generate germ-line chimeras, and bred to obtain progeny that are homozygous for the knock out gene. Such general methods of generating knock out animal have been demonstrated using mice. However, genes in other animals such as rats, guinea pigs, gerbils, hamsters, and rabbits, may also be used as long as sufficient DNA sequence data are available to make an appropriate targeting construct to knock out the gene of interest.

#The human Hox11 was originally isolated from the recurrent t(10;14)(q24;q11) chromosomal breakpoint found in human T cell acute lymphoblastic leukemia (ALL). The human cDNA Hox11 gene sequence and the corresponding amino acid sequence of the encoded open reading frame (ORF) are shown in Sequence ID Nos. 8 and 2, respectively. Probes were generated from the human cDNA sequence and used to isolate the murine genomic Hox11 gene sequence from a murine Balb-c genomic DNA library consisting of MboI partially-digested genomic DNA cloned into the bacteriophage lambda-based EMBL3 cloning vector (Sambrook et al., Molecular Cloning: A Laboratory Manual, second ed., page 2.33, Cold Spring Harbor Laboratory Press 1989).