Compositions and methods for increasing animal size growth rate Number:7,435,591 from the United States Patent and Trademark Office (PTO) owispatent The present invention relates to germ line and somatic cells comprising a mutant p27.sub.kip1 protein lacking a Cdk2 phosphorylation site. Also provided are transgenic animals and methods of making such transgenic animals which have increased size and/or growth rate.<H Compositions, increasing, Compositions, an, 7435591.html, Patent, pto, 7435591

Title: Compositions and methods for increasing animal size growth rate

Abstract: The present invention relates to germ line and somatic cells comprising a mutant p27.sub.kip1 protein lacking a Cdk2 phosphorylation site. Also provided are transgenic animals and methods of making such transgenic animals which have increased size and/or growth rate.

Patent Number: 7,435,591 Issued on 10/14/2008 to Malek, et al.

Inventors: Malek; Nisar P. (Hanover, DE), Roberts; James M. (Seattle, WA)
Assignee: Fred Hutchinson Cancer Research Center (Seattle, WA)

Appl. No.: 10/502,001

Filed: January 27, 2003

PCT Filed: January 27, 2003

PCT No.: PCT/US03/02423

371(c)(1),(2),(4) Date: July 19, 2004

PCT Pub. No.: WO03/063581

PCT Pub. Date: August 07, 2003

Related U.S. Patent Documents


Application Number	Filing Date	Patent Number	Issue Date
60352391	Jan., 2002

Current U.S. Class: 435/354 ; 435/320.1; 435/375; 435/455; 435/463

Current International Class: C12N 5/06 (20060101); C12N 15/00 (20060101); C12N 15/87 (20060101); C12N 5/00 (20060101); C12N 5/02 (20060101); C12N 5/10 (20060101)

Field of Search: 435/354,375,455,320.1,463

References Cited [Referenced By]

U.S. Patent Documents


5958769	September 1999	Roberts et al.
6242575	June 2001	Massague et al.

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Primary Examiner: Bertoglio; Valarie
Attorney, Agent or Firm: Townsend and Towsend and Crew LLP

Government Interests

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

This work was supported by a grant from the National Institutes of Health (Grant No. CA-67893. The government may have certain rights in the invention.

Parent Case Text

RELATED APPLICATIONS

This is a U.S. National Stage Application under 35 U.S.C. .sctn.371 of International Application No. PCT/US03/02423, filed on Jan. 27, 2003, which claims the benefit of U.S. Provisional Patent Application Ser. No. 60/352,391, filed on Jan. 28, 2002, the disclosures of which are incorporated herein by reference.

Claims

What is claimed is:

1. An isolated transgenic somatic or embryonic stem cell having a mutant p27.sup.Kip1 gene lacking a Cdk2 phosphorylation site integrated at the endogenous p27.sup.Kip1 gene such that there is a loss of endogenous wildtype p27.sup.Kip1 activity, wherein the mutant p27.sup.Kip1 gene encodes a mutant p27.sup.Kip1 polypeptide having a longer half-life in S phase than wildtype p27.sup.Kip1 polypeptide.

2. The transgenic cell of claim 1, wherein the mutant p27.sup.Kip1 polypeptide inhibits Cdk2 in vitro kinase activity.

3. The transgenic cell of claim 1, wherein the mutant p27.sup.Kip1 polypeptide is p27.sup.T187A.

4. An isolated transgenic mouse cell having a mutant p27.sup.Kip1 gene lacking a Cdk2 phosphorylation site integrated at the endogenous p27.sup.Kip1 gene such that there is a loss of endogenous wildtype p27.sup.Kip1 activity, wherein the mutant p27.sup.Kip1 gene encodes a mutant p27Kip1polypeptide having a longer half-life in S phase than wild-type p27.sup.Kip11 polypeptide.

5. The isolated transgenic mouse cell of claim 4, wherein the cell is a somatic cell, a germ cell, a fertilized egg, or an embryonic stem cell.

6. The isolated transgenic mouse cell of claim 5, wherein the germ cell is a an oocyte, primordial germ cell, sperm cell, or spermatocyte.

Description

BACKGROUND OF THE INVENTION

Animal cells have both a proliferating phase and a quiescent phase. Cells can shift from the proliferating phase to the quiescent phase during a brief window in the cell cycle. Depending on their position in the cell cycle, cells deprived of mitogens such as those present in serum can undergo immediate cell cycle arrest, or they can complete the current mitotic cycle and arrest in the next cell cycle. The transition from mitogen-dependence to mitogen-independence occurs in mid- to late-G1 phase of the cell cycle. Anti-mitogenic signals can cause the cell cycle to arrest at a kinetically common point. In particular, in early G1, cells can exit the cell cycle. Cell cycle commitment (autonomy from mitogenic signals) occurs in mid-G1.

The transition of cells through G1 and entry into S phase requires the action of cyclin-dependent kinases (Cdks). Growth inhibitory signals have been shown to prevent activation of these Cdks during G1 (Serrano et al., Nature 366:704-07 (1993); Hannon and Beach, Nature 371:257-61 (1994); Xiong et al., Nature 366:701-04 (1993); Polyak et al., Cell 78:59-66 (1994); Lee et al., Genes & Development 9:639-49 (1995); Koff et al., Science 260:536-39 (1993)). The catalytic activity of Cdks is known to be regulated by two general mechanisms: protein phosphorylation and association with regulatory subunits (Gould et al., EMBO J. 10:3297-309 (1991); Solomon et al., EMBO J. 12:3133-42 (1993); Solomon et al., Mol. Biol. Cell 3:13-27 (1992); Jeffrey et al., Nature 376:313-20 (1995); Morgan, Nature 374:131-34 (1995)). Among the regulatory subunits, the association of Cdks with inhibitory CKI subunits (Cyclin-dependent Kinase Inhibitors) has been most closely correlated with the effect of mitogen depletion on cell proliferation and Cdk activity.

The CKI directly implicated in mitogen-dependent Cdk regulation is p27.sup.Kip1 (Polyak et al., Cell 78:59-66 (1994); Toyoshima and Hunter, Cell 78:67-77 (1994)). Wildtype p27.sup.Kip1 protein accumulates to high levels in quiescent cells, and is rapidly destroyed after quiescent cells are re-stimulated with specific mitogens (Nourse et al., Nature 372:570-73 (1994); Kato et al., Cell 79:487-96 (1994)). The destruction of p27.sup.Kip1 is controlled by phosphorylation of p27.sup.Kip1 at threonine 187 (T187). T187 is phosphorylated by Cdk2 to create a binding site for a Skp2-containing ubiquitin-protein ligase known as the Skp1-cullin-F-box protein ligase (SCF) (Feldman et al., Cell 91:221-30 (1997); Bai et al., Cell 86:263-74 (1996); Skowyra et al., Cell 91:209-19 (1997)). Ubiquitination of p27.sup.Kip1 by the SCF then results in p27.sup.Kip1 degradation by the proteosome (Sutterluty et al., Nature Cell Biol. 1:207-14 (1999); Rolfe et al., J. Mol. Med. 75:5-17 (1997); Carrano et al., Nature Cell Biol. 1:193-99 (1999); Tsvetkov et al., Curr. Biol. 9:661-64 (1999)).

The destruction of p27.sup.Kip1 was thought to be required for entry into S phase. Moreover, constitutive expression of p27.sup.Kip1 in cultured cells causes the cell cycle to arrest in G1 (Polyak, supra; Toyoshima and Hunter, supra). Thus, based on these observations, it was expected that cells harboring a null allele of p27.sup.Kip1 would arrest G1. It was surprising, therefore, that animals harboring a null allele of the p27.sup.Kip1 gene survived. Indeed, such animals were larger than normal (increased animal size) and without apparent gross morphologic abnormalities. (Fero et al., Cell 85:733-44 (1996); U.S. Pat. No. 5,958,769; the disclosures of which are incorporated by reference herein.) The advantages of producing larger animals are readily apparent, and include increase meat, milk and/or egg production.

Decreased levels of p27.sup.Kip1 in animals, however, cause certain minor defects, such as an ovulatory defect, and resulting female sterility, increased pituitary tumorigenesis and disrupted retinal architecture. (Fero et al., supra.) These defects can interfere with some uses of such animals. Thus, there is a need for alternative mutant alleles of p27.sup.Kip1, and of methods of using such mutant alleles, that promote increased animal size or growth rate without these side effects.

SUMMARY OF THE INVENTION

The present invention relates to nucleic acids encoding a mutant p27.sup.Kip1 protein that lacks a Cdk2 phosphorylation site, and to cells harboring mutant p27.sup.Kip1 genes. In related aspects, transgenic cells and transgenic animals are provided that have one or more mutant p27.sup.Kip1 genes encoding protein that lacks a Cdk2 phosphorylation site.

In one aspect, isolated transgenic cells are provided comprising a mutant p27.sup.Kip1 gene lacking a Cdk2 phosphorylation site. The mutant p27.sup.Kip1 gene encodes a mutant p27.sup.Kip1 protein having a longer half-life in S phase than wildtype p27.sup.Kip1 polypeptide. In certain embodiments, the mutant p27.sup.Kip1 polypeptide can inhibit Cdk2 in vitro kinase activity. In an embodiment, the mutant p27.sup.Kip1 polypeptide is p27.sup.T187A.

The mutant p27.sup.Kip1 gene can be located, for example, at an endogenous p27.sup.Kip1 locus; the endogenous locus can be heterozygous or homozygous for the mutant p27.sup.Kip1 gene. The transgenic cell can be, for example, a primordial germ cell, oocyte, egg, spermatocyte, sperm cell, fertilized egg, zygote, embryonic stem cell, or somatic cell. The transgenic cell can also be progeny of any of these.

In another aspect, non-human, transgenic animals are provided which comprise a nucleic acid sequence encoding a mutant p27.sup.Kip1 protein lacking a Cdk2 phosphorylation site. In an embodiment, the mutant p27.sup.Kip1 protein is p27.sup.T187A. The transgenic animal can be, for example, a primate, mammal, bovine, porcine, ovine, equine, avian, rodent, fowl, piscine, or crustacean. In certain embodiments, the transgenic animal is a farm animal, such as a chicken, cow, bull, horse, pig, sheep, goose or duck.

In a related aspect, a transgenic, non-human animal is provided whose genome comprises a p27.sup.Kip1 gene and expresses a mutant p27.sup.Kip1 polypeptide having a longer half-life in S phase than wildtype p27 polypeptide. Expression of the mutant p27.sup.Kip1 polypeptide results in increased size or growth rate of the animal. The transgenic animal, can be, for example, a primate, mammal, bovine, porcine, ovine, equine, avian, rodent, fowl, piscine, or crustacean. In certain embodiments, the transgenic animal is a farm animal, such as a chicken, cow, bull, horse, pig, sheep, goose or duck.

Methods of increasing the size or growth rate of a non-human, transgenic animal are also provided. Such methods generally include stably introducing into a genome of an animal cell a mutant p27.sup.Kip1 gene lacking a Cdk2 phosphorylation site; and producing an animal from the animal cell. In an embodiment, the method further includes transferring a nucleus from the animal cell into a second cell from which an animal can be reconstituted; and allowing the second cell to develop into an immature animal. The immature animal typically is larger than an immature animal not having the mutant p27.sup.Kip1 gene. The second cell, can be, for example, an enucleated fertilized egg.

In another embodiment, the mutant p27.sup.Kip1 gene can be homologously integrated at an endogenous p27.sup.Kip1 locus in the animal cell. The mutant p27.sup.Kip1 gene can be homologous or heterologous to the animal cell, and can be integrated at an endogenous p27.sup.Kip1 locus or at a non-p27.sup.Kip1 locus. The mutant p27.sup.Kip1 gene can encode, for example, p27.sup.T187A protein.

The animal cell can be, for example, a germ cell, a totipotent cell, a stem cell, an embryonic stem cell, a pluripotent stem cell, a fetal cell, a primordial germ cell, an oocyte, an egg, a spermatocyte, a sperm cell, a fertilized egg, a zygote, a blastomere, or a somatic cell. The animal cell can be a vertebrate cell, such as, for example, from a primate, mammal, bovine, porcine, ovine, equine, avian, rodent, fowl, piscine, or crustacean. Exemplary animals include a chicken, hen, rooster, cow, bull, duck or goose.

Mutant genes can be introduced into cells by electroporation, microinjection, lipofection, transfection, biolistics, and the like. The mutant p27.sup.Kip1 genes can be introduced alone or as part of an expression cassette that includes, for example, a heterologous promoter operably associated with an open reading frame encoding a mutant p27.sup.Kip1 gene operably associated with a polyadenylation sequence. The expression cassette can also optionally include a selectable marker, such as the neomycin resistance gene. In an embodiment, the expression cassette can be introduced into a cell using a viral vector.

In another aspect, a method for making a large fowl is provided. The method includes introducing a mutant p27.sup.Kip1 gene lacking a Cdk2 phosphorylation site into the genome of a fowl cell by contacting in vivo a blastodermal cell of a fertilized cell with the mutant p27.sup.Kip1 gene, wherein the p27.sup.Kip1 gene is introduced directly into the germinal disk of the egg. Suitable fowl cells include those from chickens, ostriches, emus, turkeys, ducks, geese, quail, parrots, parakeets, cockatoos or cockatiels.

A further understanding of the present invention will be obtained by reference to the following description that sets forth illustrative embodiments.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

The present invention relates to nucleic acids encoding a mutant p27.sup.Kip1 protein that lacks a Cdk2 phosphorylation site and to cells harboring mutant p27.sup.Kip1 genes. In related aspects, transgenic cells and transgenic animals are provided that have one or more mutant p27.sup.Kip1 genes encoding protein that lacks a Cdk2 phosphorylation site.

The p27.sup.Kip1 protein is phosphorylated by a Cdk at a phosphorylation site to create a recognition sequence for a SCF (e.g., Cdk2). The absence or alteration of the Cdk2 phosphorylation site in p27.sup.Kip1 reduces or eliminates phosphorylation. Although the mutant p27.sup.Kip1 polypeptide is degraded in mid-G1 by the same pathway that degrades wildtype p27, the mutant p27.sup.Kip1 polypeptide has a longer half life in the S phase of the cell cycle as compared with wildtype p27.sup.Kip1 polypeptide. Yet, the mutant p27.sup.Kip1 protein can retain other functions, such as the ability to inhibit Cdk2 in vitro kinase activity.

In one aspect of the invention, isolated mutant p27.sup.Kip1 genes are provided for introduction into animal cells. (The term "isolated" refers to a molecule, such as a nucleic acid, or cell, that has been removed from its natural cellular environment. For example, an isolated nucleic acid is typically at least partially purified from other cellular nucleic acids, polypeptides and other constituents.) The mutant p27.sup.Kip1 gene encodes a p27.sup.Kip1 polypeptide lacking a Cdk2 phosphorylation site, such that less than about 10% of the mutant p27.sup.Kip1 polypeptide is phosphorylated at the Cdk2 phosphorylation site. In certain embodiments, phosphorylation at the Cdk2 phosphorylation site is less than about 5%, or less than about 1%.

The Cdk2 phosphorylation site can be defined by the following four amino acid consensus sequence: (Ser/Thr)ProXaa(Lys/Arg) or the consensus sequence (Ser/Thr)ProXaa(Lys/Arg/His/Pro), wherein Xaa can be any amino acid residue. (See, e.g., Holmes and Solomon, J. Biol. Chem. 271:25240-46 (1996).) Phosphorylation can be inhibited by substitutions, insertions and/or deletions (e.g., 1-3 amino acid insertions or deletions).

Referring to Table 1, the Cdk2 phosphorylation site, including the phosphorylated residue, is generally conserved in p27.sup.Kip1 polypeptides. As shown in the table, an asterisk indicates the position of a conserved threonine at position 187 of the human Cdk2 phosphorylation site. As used herein, this conserved threonine is referred to as threonine 187 (T187), although the skilled artisan will appreciate that this conserved residue may not be at position 187 in all p27.sup.Kip1 polypeptides. For example, in the mouse, hamster and rat polypeptide sequences, the conserved, phosphorylated residue is at position 186, although it is identifiable by sequence alignment and by biochemical analysis, as discussed in the Examples (infra). Thus, the terms "T187," "T187A" and position "187" are merely

TABLE-US-00001 TABLE 1 * Consensus 151 IRKRPATDDSSTQNKRANRTEENVSDGSPNAGSVEQTPKKPGLRRRQT 198 (SEQ ID NO:4) Genbank Species Residues 7769665 Human 151 ........................L....................... 198 SE- Q ID NO:5) 4757962 Human 151 ................................................ 198 SE- Q ID NO:6) 12805035 Human 151 ................................................ 198 SE- Q ID NO:6) 2135228 Human 151 ................................................ 198 SE- Q ID NO:6) 3913222 Cat 151 ...........P.................................... 198 SEQ ID NO:7) 13429931 Pig 151 ...........P..................SA................ 198 SEQ ID NO:8) 6753386 Mouse 151 M.....AE...S....................T............Q 196 SEQ ID NO:9) 2493565 Hamster 151 M.....A....S................L................H.. 198 - SEQ ID NO:10) 2102649 Rat 151 M.....AE...S....................T............Q 196 SEQ ID NO:11) 2281010 Rat 151 M.....AE...S.....S..............T............Q 196 SEQ ID NO:12)

shorthand for this conserved threonine residue position and not to be limited to amino acid 187 of a p27.sup.Kip1 polypeptide, or the corresponding codon in a p27.sup.Kip1 gene.

A Cdk2 phosphorylation site in a p27.sup.Kip1 polypeptide can be identified, for example, by biochemical analysis. (See, e.g., Holmes and Solomon, supra.) A Cdk2 phosphorylation site in a p27.sup.Kip1 gene and/or polypeptide sequence also can be identified by alignment with known p27.sup.Kip1 gene and/or polypeptide sequences. For example, an alignment can be performed by the local homology algorithm of Smith and Waterman (Adv. Appl. Math. 2:482 (1981), which is incorporated by reference herein in its entirety), by the homology alignment algorithm of Needleman and Wunsch (J. Mol. Biol. 48:443-53 (1970), which is incorporated by reference herein in its entirety), by the search for similarity method of Pearson and Lipman (Proc. Natl. Acad. Sci. USA 85:2444-48 (1988), which is incorporated by reference herein in its entirety), by computerized implementations of these algorithms (e.g., GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package (Accelrys), or by visual inspection.

One example of a useful algorithm is PILEUP. PILEUP creates a multiple sequence alignment from a group of related sequences using progressive, pairwise alignments to show the percent sequence identity. It also plots a tree or dendogram showing the clustering relationships used to create the alignment. PILEUP uses a simplification of the progressive alignment method of Feng and Doolittle (J. Mol. Evol. 25:351-60 (1987), which is incorporated by reference herein in its entirety). The method used is similar to the method described by Higgins and Sharp (Comput. Appl. Biosci. 5:151-53 (1989), which is incorporated by reference herein in its entirety). The program can align up to 300 sequences, each of a maximum length of 5,000 nucleotides or amino acids. The multiple alignment procedure begins with the pairwise alignment of the two most similar sequences, producing a cluster of two aligned sequences. This cluster is then aligned to the next most related sequence or cluster of aligned sequences. Two clusters of sequences are aligned by a simple extension of the pairwise alignment of two individual sequences. The final alignment is achieved by a series of progressive, pairwise alignments. The program is run by designating specific sequences and their amino acid or nucleotide coordinates for regions of sequence comparison and by designating the program parameters. For example, a reference sequence can be compared to other test sequences to determine the percent sequence identity relationship using the following parameters: default gap weight (3.00), default gap length weight (0.10), and weighted end gaps.

Another example of an algorithm that is suitable for aligning sequences, and for determining percent sequence identity and sequence similarity, is the BLAST algorithm, which is described by Altschul et al. (J. Mol. Biol. 215:403-410 (1990), which is incorporated by reference herein in its entirety). (See also Zhang et al., Nucleic Acid Res. 26:3986-90 (1998; Altschul, et al., Nucleic Acid Res. 25:3389-402 (1997), which are incorporated by reference herein in their entirety). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Extension of the word hits in each direction is halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLAST program uses as default parameters a wordlength (W) of 11, the BLOSUM62 scoring matrix (see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915-19 (1992), which is incorporated by reference herein in its entirety) alignments (B) of 50, expectation (E) of 10, M=5, N=-4, and a comparison of both strands. The skilled artisan will appreciate, however, that other parameters can be used.

The isolated, mutant p27.sup.Kip1 genes can be, for example, genomic DNA, cDNA, RNA, mRNA, and the like, as well as fragments of any of these. The mutant p27.sup.Kip1 genes can be polynucleotides or nucleic acids or other polymers composed of a multiplicity of nucleotide units (ribonucleotide or deoxyribonucleotide or related structural variants) linked via phosphodiester bonds. Mutant genes can be of substantially any length, typically from about twelve (12) nucleotides to about 10.sup.9 nucleotides or larger, that do not encode a Cdk2 phosphorylation site. In one embodiment, a fragment of a mutant p27.sup.Kip1 gene has at least 50 contiguous nucleotides; in other embodiments, the fragment of the mutant p27.sup.Kip1 gene is at least 100 nucleotides, at least 200 nucleotides, at least 500 nucleotides, at least 1000 nucleotides, or more of the gene. In related embodiments, the mutant p27.sup.Kip1 gene is at least an exon, a cDNA, or a fall length genomic p27.sup.Kip1 gene, lacking a Cdk2 phosphorylation site.

Mutant p27.sup.Kip1 genes also include derivatives, such as those based on all possible codon choices for an amino acid(s) that, when expressed from a mutant p27.sup.Kip1 gene, results in the expression of a mutant protein in which Cdk-mediated phosphorylation is inhibited. At amino acid positions outside the Cdk2 phosphorylation site, mutant p27.sup.Kip1 gene derivatives can include those based on all possible codon choices for the same amino acid and codon choices based on conservative amino acid substitutions. For example, the following six groups each contain amino acids that are conservative substitutions for one another: 1) Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W). (See also Creighton, Proteins, W. H. Freeman and Company (1984).) In addition, individual substitutions, deletions or additions that alter, add or delete a single amino acid or a small percentage of amino acids in an encoded sequence are also "conservative substitutions."

In certain embodiments, mutant p27.sup.Kip1 genes be synthesized, or chemically or biochemically modified (e.g., can contain non-natural or derivatized nucleotide bases). Such modifications include, for example, labels, methylation, substitutions of one or more of the naturally-occurring nucleotides with an analog, internucleotide modifications such as uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoamidates, carbamates, and the like), charged linkages (e.g. phosphorothioates, phosphorodithioates, and the like), pendent moieties (e.g., polypeptides), intercalators (e.g., acridine, psoralen, and the like), chelators, alkylators, and modified linkages (e.g., alpha anomeric nucleic acids, and the like).

The mutant p27.sup.Kip1 gene(s) can be homologous or heterologous to the cell or the animal. As used herein, the term "homologous" p27.sup.Kip1 gene refers to a p27.sup.Kip1 gene derived from the same species as the cell or animal. A "heterologous" p27.sup.Kip1 gene refers to a p27.sup.Kip1 gene from a different species. For example, if the animal is a chicken, a homologous mutant p27.sup.Kip1 gene is derived from a chicken p27.sup.kip1 gene, while a heterologous mutant p27.sup.Kip1 gene is derived, for example, from a mouse p27.sup.Kip1 gene.

The mutant p27.sup.Kip1 gene can be prepared by, for example, mutagenizing a wild-type p27.sup.Kip1 gene at one or more positions in the Cdk2 phosphorylation site. In various embodiments, the p27.sup.Kip1 gene is human, primate, mammalian, avian, porcine, ovine, bovine, fowl, rodent, fish, crustacean, and the like. In specific embodiments, the p27.sup.Kip1 is from a sheep, goat, horse, cow, bull, pig, rabbit, guinea pig, hamster, rat, gerbil, mouse, chicken, ostrich, emu, turkey, duck, goose, quail, parrot, parakeet, cockatoo, cockatiel, trout, cod, salmon, crab, king crab, lobster, shrimp, and the like. p27.sup.Kip1 gene sequences are disclosed for example, in the GenBank database under accession numbers gi|7769665|, gi|4757962|, gi|12805035|, gi|2135228|, gi|3913222|, gi|13429931|, gi|6753386|, gi|2493565|, gi|2102649|, and gi|2281010|, which are incorporated by reference herein in their entirety. p27.sup.Kip1 polypeptide sequences are disclosed, for example, in the GenBank database under accession numbers AAF69497.1, NP.sub.--004055.1, AAH01971.1, I52718, O19001, BAB39725.1, NP.sub.--034005.1, Q60439, BAA19960.1, and BAA21561.1 (the disclosures of which are incorporated by reference herein in their entirety).

p27.sup.Kip1 genes can be readily isolated by methods known to the skilled artisan. (See generally Sambrook et al., Molecular Cloning, A Laboratory Manual, 3rd ed., Cold Spring Harbor Publish., Cold Spring Harbor, N.Y. (2001); Ausubel et al., Current Protocols in Molecular Biology, 4th ed., John Wiley and Sons, New York (1999); which are incorporated by reference herein in their entirety.) Specific embodiments for the isolation of p27.sup.Kip1 genes, presented as example but not by way of limitation, are described below.

p27.sup.Kip1 genes can be isolated, for example, by polymerase chain reaction (PCR) to amplify the p27.sup.Kip1 gene, or a portion thereof, from a genomic or cDNA library. Oligonucleotide primers representing known p27.sup.Kip1 sequences can be used as primers in PCR. In a typical embodiment, the oligonucleotide primers represent at least a fragment of conserved segments of identity between p27.sup.Kip1 genes of different species. Synthetic oligonucleotides can be utilized as primers to amplify particular oligonucleotides within a p27.sup.Kip1 gene by PCR sequences from any suitable source (e.g., RNA or DNA), typically a cDNA library or mRNA of potential interest. PCR can be carried out, for example, by use of a Perkin-Elmer Cetus thermal cycler and Taq polymerase (Gene Amp). Degenerate primers can be designed for use in the PCR reactions. For example, the CODEHOP strategy of Rose et al. (Nucl. Acids Res. 26:1628-35 (1998), which is incorporated by reference herein in its entirety) can be used to design degenerate PCR primers using multiply-aligned sequences as a reference. Methods for performing PCR and related methods are well known in the art. (See, e.g., U.S. Pat. Nos. 4,683,202; 4,683,195 and 4,800,159; Innis et al., PCR Protocols: A Guide to Methods and Applications, Academic Press, Inc., San Diego, Calif. (1989); Innis et al., PCR Applications: Protocols for Functional Genomics, Academic Press, Inc., San Diego, Calif. (1999); White (ed.), PCR Cloning Protocols: From Molecular Cloning to Genetic Engineering, Humana Press, (1996); EP 320 308; the disclosures of which are incorporated by reference herein in their entirety.)

In an embodiment, degenerate primers are used to isolate the p27.sup.Kip1 cDNA from an avian species. Avian species are known to have p27.sup.Kip1. (See Torchinsky et al., J. Neurocytol. 28:913-24 (1999).) Briefly, an alignment of multiple p27.sup.Kip1 polypeptide sequences from different animals is prepared and used to visually identify blocks of sequences having low codon degeneracy (see Rose et al. (supra)). The CODEHOP strategy of Rose et al. (supra) is used to design degenerate primers based on the blocks of low codon degeneracy. Pools of primers varying in redundancy from 2 fold to about 32 fold are prepared. A hemi-nested PCR strategy is used to amplify fragments from an avian chicken cDNA library (e.g., a chicken or hyacinth macaw library from Stratagene). Briefly, PCR is performed at 55.degree. C. using the primer pools. (See, e.g., Rose et al. (supra); Rose et al., J. Virology 71:4138-44 (1997).) PCR amplification products can be detected, for example, by agarose gel electrophoresis. The identity of the PCR amplification products can be confirmed by DNA sequence analysis. Once the identify of the PCR amplification products is confirmed, the amplification products can be used to isolate full length p27.sup.Kip1 cDNA from the avian cDNA library. (See, e.g., Sambrook et al., supra; Ausubel et al., supra.)

For expression cloning (a technique commonly known in the art), an expression library is constructed by methods known in the art. For example, mRNA is isolated, cDNA is prepared and then ligated into an expression vector (e.g., a bacteriophage derivative) such that it is capable of being expressed by the host cell into which it is then introduced. Various screening assays can then be used to select for the expressed p27.sup.Kip1 polypeptide. In one embodiment, polyclonal antibodies against a mammalian p27.sup.Kip1 polypeptide (see, e.g. U.S. Pat. No. 6,242,575; the disclosure of which is incorporated by reference herein in its entirety) are used to screen a chicken cDNA expression library (e.g., from Strategene) to identify avian p27.sup.Kip1 genes.

Alternatively, p27.sup.Kip1 genes can be isolated by hybridization using a heterologous p27.sup.Kip1 nucleic acid as a probe. For example, p27.sup.Kip1 genes can be isolated by screening a genomic or cDNA library with a p27.sup.Kip1 nucleic acid probe. Such a probe can be, for example, a portion of a p27.sup.Kip1 gene or its specific RNA, or a fragment thereof, that exhibits low codon degeneracy. Such a probe can be prepared, detectably labeled, and used to screen a library by nucleic acid hybridization (see, e.g., Benton and Davis, Science 196:180-82 (1977); Grunstein and Hogness, Proc. Natl. Acad. Sci. USA 72:3961-65 (1975); Sambrook et al., supra; Ausubel et al., supra). DNA fragments with substantial identity to the probe will hybridize and can be identified using the detectable label.

In various embodiments, hybridization screening using a heterologous p27.sup.Kip1 nucleic acid probe can assist in the isolation of p27.sup.Kip1 genes. p27.sup.Kip1 genes can be isolated, for example, from human or non-human sources, such as, for example, primato, porcine, bovine, feline, equine, canine, ovine, avian, reptilian, amphibian, piscine, and the like; and from non-vertebrate sources, such as insects, worms, nematodes, and the like. In certain embodiments, the isolated p27.sup.Kip1 gene can be from a chicken, goose, duck, lobster, rabbit, sheep, cow, bull, horse, pig, and the like.

By way of example, and not limitation, procedures using low stringency conditions are as follows: Filters containing DNA are pretreated for 6 hours at 40.degree. C. in a solution containing 35% formamide, 5.times.SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.1% polyvinylpyrrolidone (PVP), 0.1% Ficoll, 1% bovine serum albumin (BSA), and 500 .mu.g/ml denatured salmon sperm DNA. Hybridizations are carried out in the same solution with the following modifications: 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 .mu.g/ml salmon sperm DNA, 10% (wt/vol) dextran sulfate, and 5-20.times.10.sup.6 cpm .sup.32P-labeled probe. Filters are incubated in hybridization mixture for 18-20 hours at 40.degree. C., and then washed for 1.5 hours at 55.degree. C. in a solution containing 2.times.SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS. The wash solution is replaced with fresh solution and incubated an additional 1.5 hours at 60.degree. C. Filters are blotted dry and exposed for autoradiography. If necessary, filters are washed for a third time at 65-68.degree. C. and re-exposed to film. Other conditions of low stringency that can be used are well known in the art (e.g., those employed for cross-species hybridizations). (See also Shilo and Weinberg, Proc. Natl. Acad. Sci. USA 78:6789-92 (1981); Sambrook et al., supra; Ausubel et al., supra.)

Alternatively, moderate stringency conditions can be used. By way of example, and not limitation, procedures using such conditions of moderate stringency are as follows: Prehybridization of filters containing DNA is carried out for 8 hours to overnight at 55.degree. C. in buffer composed of 6.times.SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.2% Ficoll, 0.02% BSA and 500 .mu.g/ml denatured salmon sperm DNA. Filters are hybridized for 24 hours at 55.degree. C. in a prehybridization mixture containing 100 .mu.g/ml denatured salmon sperm DNA and 5-20.times.10.sup.6 cpm of .sup.32P-labeled probe. Washing of filters is done at 37.degree. C. for 1 hour in a solution containing 2.times.SSC, 0.01% PVP, 0.01% Ficoll, and 0.01% BSA.

By way of example, and not limitation, procedures using conditions of high stringency are as follows: Prehybridization of filters containing DNA is carried out for 8 hours to overnight at 65.degree. C. in buffer composed of 6.times.SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 .mu.g/ml denatured salmon sperm DNA. Filters are hybridized for 48 hours at 65.degree. C. in prehybridization mixture containing 100 .mu.g/ml denatured salmon sperm DNA and 5-20.times.10.sup.6 cpm of .sup.32P-labeled probe. Washing of filters can be performed at 65.degree. C. for 1 hour in a solution containing 2.times.SSC, 0.01% PVP, 0.01% Ficoll, and 0.01% BSA. This is followed by a wash in 0.1.times.SSC at 50.degree. C. for 45 minutes before autoradiography. Other conditions of high stringency which can be used are well known in the art. (See, e.g., Ausubel et al., supra; Sambrook et al., supra.)

Various other hybridization conditions can be used. For example, hybridization in 6.times.SSC at about 45.degree. C., followed by washing in 2.times.SSC at 50.degree. C. can be used. Alternatively, the salt concentration in the wash step can range from low stringency of about 5.times.SSC at 50.degree. C., to moderate stringency of about 2.times.SSC at 50.degree. C., to high stringency of about 0.2.times.SSC at 50.degree. C. In addition, the temperature of the wash step can be increased from low stringency conditions at room temperature, to moderately stringent conditions at about 42.degree. C., to high stringency conditions at about 65.degree. C. Other conditions include, but are not limited to, hybridizing at 68.degree. C. in 0.5M NaH.sub.2PO.sub.4 (pH7.2)/1 mM EDTA/7% SDS, or hybridization in 50% formamide/0.25M NaH.sub.2PO.sub.4 (pH7.2)/0.25 M NaCl/1 mM EDTA/7% SDS, followed by washing in 40 mM NaH.sub.2PO.sub.4 (pH7.2)/1 mM EDTA/5% SDS at 50.degree. C. or in 40 mM NaH.sub.2PO.sub.4 (pH7.2)/1 mM EDTA/1% SDS at 50.degree. C. Both temperature and salt can be varied, or alternatively, one or the other variable can remain constant while the other is changed.

Low, moderate and high stringency conditions are well known to those of skill in the art, and will vary predictably depending on the base composition of the particular nucleic acid sequence and on the specific organism from which the nucleic acid sequence is derived. For guidance regarding such conditions see, for example, Sambrook et al. (supra) and Ausubel et al. (supra).

p27.sup.Kip1 genes can also be identified, for example, by searching a genomic sequence database, such as those for Drosophila, C elegans, and the like. Such searches can be performed, for example, using the Blast search engine (Altschul et al., Nucleic Acids Res. 25:3389-402 (1997)), or other suitable sequence comparison program. Information and tools for screening genomic databases are provided, for example, at the NCBI Internet web site (http://www.ncbi.nlm.nih.gov), as well as from commercially available sources. The UniGene collection provides a non-redundant set of sequences that represent unique genes of different sequences. (See www.ncbi.nlm.nih.gov.) This collection includes well-characterized genes, as well as thousands of expressed sequence tag (EST) sequences.

The methods discussed above are not meant to limit the methods by which p27.sup.Kip1 genes can be isolated. p27.sup.Kip1 genes derived frown genomic DNA can contain regulatory and intron DNA regions in addition to coding regions; clones derived from cDNA will typically contain only exon sequences. Nucleic acids can be molecularly cloned into a suitable vector for propagation of those nucleic acids. (See, e.g., Sambrook et al., supra; Ausubel et al., supra.)

A p27.sup.Kip1 gene can be mutagenized to create a substitution, deletion and/or insertion in the Cdk2 phosphorylation site. In an exemplary embodiment, a substitution of the phosphorylated threonine or serine is made by altering the codon that codes for that residue. In other embodiments, other residues in the Cdk2 phosphorylation site can be changed or deleted. This can be accomplished, for example, by site-directed mutagenesis using the Amersham technique (Amersham mutagenesis kit, Amersham, Inc., Cleveland, Ohio) based on the methods of Taylor et al. (Nucl. Acids Res. 13:8749-84 (1985); Nucl. Acids Res. 13:8764-85 (1985)), Nakamaye and Eckstein (Nucl Acids Res. 14:9679-98 (1986)); and Dente et al. (DNA Cloning, Glover, Ed., IRL Press, pp. 791-802 (1985)); using a Promega kit (Promega Inc., Madison, Wis.); using a Biorad kit (Biorad Inc., Richmond, Calif.), based on the methods of Kunkel (Proc. Natl. Acad. Sci. USA 82:488-92 (1985); Meth. Enzymol. 154:367-82(1987); U.S. Pat. No. 4,873,192), and the like. Site directed mutagenesis can also be accomplished using PCR-based mutagenesis, such as the technique described by Zhengbin et al. (in PCR Methods and Applications, Cold Spring Harbor Laboratory Press, New York, pp. 205-207 (1992)), by Jones and Howard (BioTechniques 8:178-83 (1990); BioTechniques 10:62-66 (1991)); by Ho et al. (Gene 77:51-59 (1989)), and by Horton et al. (BioTechniques 8:528-35 (1990); Gene 77:61-68 (1989)). Other methods of mutagenizing a p27.sup.Kip1 gene to modify a Cdk2 phosphorylation site are known to the skilled artisan and are within the scope of the invention.

A mutant p27.sup.Kip1 gene can be part of an expression cassette, ie., having a promoter and a coding region encoding a mutant p27.sup.Kip1 polypeptide. The promoter can be a homologous promoter (i.e., a p27.sup.Kip1 gene promoter from the same species) or a heterologous promoter (i.e., a p27.sup.Kip1 gene promoter from a different species, or a non-p27.sup.Kip1 gene promoter) for expression of a mutant p27.sup.Kip1 coding region (i.e., lacking a Cdk2 phosphorylation site). As used herein, the term "coding region" refers to a nucleotide sequence containing a translational initiation codon followed by an ordered arrangement of codons that encode a mutant p27.sup.Kip1 protein and a translational termination codon. A "coding region" can also encode a fragment of a mutant p27.sup.Kip1 protein lacking a Cdk2 phosphorylation site. The promoter is operably or operatively associated with the coding region, whereby the promoter effects expression of the coding region.

Suitable heterologous promoters include, for example, promoters that are expressed in a wide variety of tissue types, such as, for example, the SV40 early promoter region (Benoist and Chambon, Nature 290:304-10 (1981)), the promoter contained in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto et al., Cell 22:787-97 (1980)), the herpes thymidine kinase promoter (Wagner et al., Proc. Natl. Acad. Sci. USA 78:1441-45 (1981)), the regulatory sequences of the metallothionein gene (Brinster et al., Nature 296:39-42 (1982)), the cytomegalovirus (CMV) promoter, the mouse Oct4 gene promoter (International Patent Publication No. WO 00/56932), the Mouse Moloney Leukemia Virus LTR (Miller and Buttimore, Mol. Cell. Biol. 6:2895-902 (1986), Gossen and Bujard, Proc. Natl. Acad. Sci. USA 89:5547-51 (1992); Pescini et al., Biochem. Biophys Res. Comm. 202:1664-67 (1994); ubiquitously expressed promoters such as the ROSA26 and G3BP promoters (Zambrowicz et al., Proc. Natl. Acad. Sci. USA 94:3789-94 (1997); Parker et al., Molecular and Cellular Biology 16:2561-69 (1996)); and the like.

For expression in avian species, the promoter can be, for example, lactoferrin-derived transcription regulatory sequences (International Publication No. WO 00/75300), the chicken ovalbumin promoter (Genbank Accession Nos. J00895 or M24999), the chicken lysozyme promoter (Genbank Accession Nos. J00886 or V00429), and the like. Other suitable promoters are known to those of skill in the art. In certain embodiments, an Internal Ribosomal Entry Site (IRES) can be part of a promoter system express a mutant p27.sup.Kip1 gene. Suitable polyadenylation sequences include, for example, the human beta-globin polyadenylation sequence, and the SV40 early polyadenylation sequence.

The mutant p27.sup.Kip1 gene expression cassette optionally can further include a selectable marker, such as a positively and/or negatively selectable marker. Suitable positively selectable markers can include, for example, the neomycin gene, the hygromycin gene, the hisD gene, the xanthine-guanine phosphoribosyltransferase (Gpt) gene conferring resistance to mycophenolic acid (Mulligan et al., Proc. Natl. Acad. Sci. USA 78:2072-76 (1981)), the hypoxanthine phosphoribosyl transferase (Hprt) gene, and the like. Suitable negative selection markers include, for example, the HSV thymidine kinase gene, the Hprt gene, the Gpt gene, Diphtheria toxin, Ricin toxin, cytosine deaminase, and the like. The selectable marker typically confers a phenotype for identification and isolation of cells containing an introduced mutant p27.sup.Kip1 gene.

A mutant p27.sup.Kip1 gene optionally can be part of an expression vector. Such an expression vector typically comprises an expression cassette (e.g., a promoter operably linked to a mutant p27.sup.Kip1 gene operably linked to a polyadenylation sequence), one or more origins of replication, and, optionally, one or more selectable markers (e.g., an antibiotic resistance gene and/or any of those describe above). Suitable origins of replication include, for example, the SV40 origin of replication, the colE1 origin of replication, and the like.

Suitable expression vectors can include defective or attenuated retroviral vectors or other viral vector (see, e.g., U.S. Pat. No. 4,980,286). For example, a retroviral vector, as described by Miller et al. (Meth. Enzymol. 217:581-99 (1993)) can be used. (See also Boesen et al., Biotherapy 6:291-302 (1994).) (These references are incorporated herein in their entirety.) These retroviral vectors are typically modified to delete retroviral sequences that are not necessary for packaging of the viral genome and integration into host cell DNA. The mutant p27.sup.Kip1 gene is inserted into the vector, which facilitates delivery of the gene into a cell. Lentiviral vectors can also be used. (See, e.g., Naldini et al., Science 272:263-67 (1996), incorporated by reference herein in its entirety.)

Adenoviruses can also be used as an expression vector to introduce a mutant p27.sup.Kip1 gene into cells. Adenoviruses have the advantage of being capable of infecting non-dividing cells. Adeno-associated virus (AAV) are another suitable vector. (See, e.g., Ali et al., Gene Therapy 1:367-84 (1994); U.S. Pat. Nos. 4,797,368 and 5,139,941; Walsh et al., Proc. Soc. Exp. Biol. Med. 204:289-300 (1993); Grimm et al., Human Gene Therapy 10:2445-50 (1999); the disclosures of which are incorporated by reference herein in their entirety.)

The expression cassette or vector can be used for homologous integration of a mutant p27.sup.Kip1 gene at a predetermined locus in the genome of a cell. For example, a mutant p27.sup.Kip1 gene can be homologously integrated at an endogenous p27.sup.Kip1 locus in a cell. Alternatively, a mutant p27.sup.Kip1 gene can be integrated at any other suitable locus in a cell, such as a non-essential gene locus or other non-essential genomic region. As used herein, the term "homologous recombination" refers to a process of recombination or gene conversion whereby homology regions flanking a mutant p27.sup.Kip1 gene, or a portion thereof (e.g. the nucleic acid sequence encoding a Cdk2 phosphorylation site), replace corresponding chromosomal sequences in the genome of the cell.

Homologous recombination can occur by, for example, double-crossover replacement recombination, in which homologous recombination (e.g., strand exchange, strand pairing, strand scission, and strand ligation) occurs between homology regions in an expression vector or expression construct and chromosomal sequences in a cell. The homology regions are generally used in the same orientation (e.g., the upstream direction (5' relative to the direction of transcription) is the same for each homology region) to avoid rearrangements. Double-crossover replacement recombination thus can be used to insert a mutant p27.sup.Kip1 gene, or a portion thereof, into an endogenous gene locus. In certain embodiments, the homology regions are from an endogenous p27.sup.Kip1 gene, and the mutant p27.sup.Kip1 gene or a fragment thereof, integrates at an endogenous p27.sup.Kip1 gene locus. Alternatively, the homologous regions are from a different locus, and the mutant p27.sup.Kip1 gene is integrated at that locus.

Suitable "targeting constructs" for homologous integration of a mutant p27.sup.Kip1 gene include, for example, those disclosed in U.S. Pat. Nos. 5,631,153; 5,627,059; 5,487,992; 5,464,764; and 6,204,061 (the disclosures of which are incorporated by reference herein in their entirety). Targeting constructs can be, for example, a targeting construct for single-crossover integration, or "hit-and-run" targeting, which has only a single homology region linked to a mutant p27.sup.Kip1 gene or gene fragment. Alternatively, the targeting construct can have two homology regions, each flanking a mutant p27.sup.Kip1 gene or gene fragment. For example, a targeting construct can comprise, in order: (1) a first homology region having a sequence substantially identical to a sequence of a portion of an endogenous gene locus, (2) a mutant p27.sup.Kip1 gene or a fragment thereof, and (3) a second homology region having a sequence substantially identical to a different portion of the endogenous gene locus. In certain embodiments, the targeting construct further comprises a negatively selectable marker (e.g., Diphtheria toxin gene with the PGK promoter driving transcription) linked to an outer end of a homology region. Such a targeting construct can optionally further include a positively selectable marker disposed between the first and second homology regions. The homology regions typically range from between about 50 base pairs to about several tens of kilobases. In some embodiments, targeting constructs are generally at least about 250 nucleotides, at least about 500 nucleotides, typically at least about 1000 to about 6000 nucleotides, or longer.

The homology region(s) can be selected at the discretion of the practitioner on the basis of the sequence composition and complexity of the gene locus and guidance provided in the art (see, e.g., Hasty et al., Mol. Cell. Biol. 11:5586-91 (1991); Shulman et al., Mol. Cell. Biol. 10:4466-72 (1990), which are incorporated herein by reference in their entirety). Targeting constructs are generally double-stranded DNA molecules; most are typically linear. General principles regarding the construction of targeting constructs and selection methods are reviewed in Bradley et al. (Bio/Technology 10:534-39 (1992), incorporated herein by reference in its entirety). (See also Capecchi, Science 244:1288-92 (1989); incorporated herein by reference in its entirety.)

In another aspect, transgenic cells comprising one or more mutant p27.sup.Kip1 genes are provided. As used herein, the term "transgenic cells" refers to a human or non-human cell comprising one or more mutant p27.sup.Kip1 genes. A transgenic cells can be, for example, from a human, primate, mammal, avian, porcine, ovine, bovine, feline, canine, fowl, rodent, fish, crustacean, and the like. In specific embodiments, the transgenic cells can be from a sheep, goat, horse, cow, bull, pig, rabbit, guinea pig, hamster, rat, gerbil, mouse, chicken, ostrich, emu, turkey, duck, goose, quail, parrot, parakeet, cockatoo, cockatiel, trout, cod, salmon, crab, king crab, lobster, or shrimp.

Mutant p27.sup.Kip1 genes can be introduced into target cells, such as, for example, pluripotent or totipotent cells such as embryonic stem (ES) cells (e.g., murine embryonal stem cells or human embryonic stem cells) or other stem cells (e.g., adult stem cells); germ cells (e.g., primordial germ cells, oocytes, eggs, spermatocytes, or sperm cells); fertilized eggs; fetal or adult somatic cells, either differentiated or undifferentiated (e.g., thymocytes, fibroblasts, keratinocytes, brain, muscle, liver, lung, bone marrow, heart, neuron, gastrointestinal, kidney, spleen, or epithelial cells); and the like. In certain embodiments, the mutant p27.sup.Kip1 gene can be introduced into embryonic stem cells or germ cells.

Suitable transgenic cells can also include "cell lines," which refers to individual cells, harvested cells, and cultures containing the cells derived from cells of the cell line referred to. A cell line is said to be "continuous," "immortal," or "stable" if the line remains viable over a prolonged time, typically at least about six months. Suitable transgenic cells can also include primary cells. Primary cells include cells that are obtained directly from an organism or that are present within an organism, and cells that are obtained from these sources and grown in culture, but are not capable of continuous (e.g., many generations)