Multicellular living organisms and unmodified parts thereof and – Method of using a transgenic nonhuman animal in an in vivo...
Reexamination Certificate
1999-05-28
2004-05-04
Shukla, Ram R. (Department: 1632)
Multicellular living organisms and unmodified parts thereof and
Method of using a transgenic nonhuman animal in an in vivo...
C800S013000, C800S008000
Reexamination Certificate
active
06730820
ABSTRACT:
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
To be determined.
BACKGROUND OF THE INVENTION
Cell migration, particularly migration of cancerous cells and nerve cells, is not well understood, nor are the factors that affect cell migration and tissue shaping in vivo. There is a need in the art to identify and exploit such factors, including but not limited to those involved in normal or abnormal organogenesis. The art also lacks efficient systems for evaluating therapeutic modulators of such functions in vivo and lacks diagnostic methods for assessing the ability of a cell or cell mass to migrate in vivo.
Organogenesis processes in vertebrates proceed in a manner similar to those observed in the common laboratory nematode
C. elegans
. As such, the generation of
C. elegans
gonadal structures can serve as a simple system for investigating developmental morphogenetic processes shared by higher and lower organisms.
In one common morphogenetic process, a tissue bud extends to form an elongate tube with a proximal to distal axis. An emerging theme in bud extension is the presence of specialized regulatory cells at the bud tip that govern elongation. In vertebrate development, this process is seen in extension of the limb (Johnson and Tabin, 1997; Martin, 1998), ureter (Vainio and Muller, 1997), and lung branches (Hogan, 1998). In the
C. elegans
gonad, long “arms” develop by elongation of buds originating from a gonadal primordium. Each gonadal arm possesses a single “leader cell” that serves this regulatory role (Kimble and White, 1981). The biology of distal tip cell migration during gonadogenesis is known to one skilled in the art of
C. elegans
developmental biology. Indeed, the
C. elegans
gonadal leader cells are among the best defined cells that regulate bud elongation, and therefore serve as a paradigm for investigating this common morphogenetic process.
A second common morphogenetic process of organogenesis is the formation of a complex, differentiated epithelial tube. Formation of a complex epithelial tube can involve an initial condensation of mesenchymal cells, followed by epithelialization, lumen formation, and differentiation into modular units. Vertebrate examples include the kidney tubules (Vainio and Muller, 1997) and heart tube (Fishman and Olson, 1997). Similarly, during
C. elegans
gonadogenesis, cells coalesce to form a compact larval structure called the somatic gonadal primordium (SGP). Following formation of this primordium, cell division and differentiation are accompanied by epithelialization and lumen formation to form a complex tube composed of distinct modular units: the uterus, spermathecae and sheaths in hermaphrodites, and the seminal vesicle and vas deferens in males (Kimble and Hirsh, 1979).
Previous studies have identified several genes in
C. elegans
that influence gonadal morphogenesis. One group of such genes includes unc-5, unc-6, and unc-40, which control the direction of leader cell migration (Hedgecock et al, 1990). Normally, leader cells migrate in one direction, then move dorsally, and finally move in the opposite direction to generate a reflexed gonadal arm. In the absence of unc-5, unc-6, or unc-40, the leader cells fail to turn dorsally. Another gene, ced-5, causes the leader cell to makes extra turns or stop prematurely (Wu and Horvitz, 1998). Therefore, in these mutants, the leader cells migrate, but do not navigate correctly, which results in a failure of the gonadal arms to acquire their normal U-shape. In addition to these genes, others are required for specification of cell fates and also influence morphogenesis (lin-12: Greenwald et al., 1983, Newman et al., 1995; lin-17: Sternberg and Horvitz, 1988; lag-2: Lambie and Kimble, 1991; ceh-18: Greenstein et al., 1994, Rose et al., 1997; lin-26: den Boer et al., 1998).
A known
C. elegans
genetic locus, gon-1, defined by one or more mutants, is essential for extension of gonadal germline arms, but is not responsible for signaling the germline to proliferate. In
C. elegans
hermaphrodites, GON-1 is required for migration of two distal tip cells to produce two elongated tubes, whereas in males, gon-1 activity is required for migration of a single linker cell to produce a single elongated tube. In gon-1 mutant hermaphrodites, the leader cells are born normally in the somatic gonadal cell lineage and function normally to promote germline proliferation, but they fail to migrate and do not support arm extension. Similarly in males, the leader cell does not move and no arm extension occurs. The gon-1 locus has not heretofore been mapped with particularity to a nucleic acid coding sequence.
Clarification of the genetic basis for
C. elegans
gon-1 activity would permit one to apply molecular tools to the study of cell migration in a convenient system. It would be particularly advantageous to find that the gon-1 locus encodes a protein having structural relationship to proteins of species that are not readily studied in the laboratory, since one would be able to evaluate those proteins in the convenient
C. elegans
system. Such a system would also provide a means for evaluating agents that can modulate the activity of such genes and proteins and would both facilitate understanding the factors involved in cell migration.
BRIEF SUMMARY OF THE INVENTION
In one aspect, the invention can be an isolated polynucleotide coding sequence that encodes a protein the includes both a metalloprotease domain and at least one thrombospodin type 1 domain, where the protein can direct either cell migration or tissue shaping in an analytical system in a target organism as disclosed herein. In another aspect, the invention can also be a variant of the isolated polynucleotide coding sequence that encodes a protein that shares at least 20%, more preferably 50%, still more preferably 70% and most preferably 80% amino acid sequence identity (using GCG Pileup program) with any of the foregoing in the metalloprotease and thrombospondin type 1 domains while also comprising the amino acids of those domains known to those skilled in the art to be required for protein activity. A suitable variant polynucleotide can hybridize under stringent hybridization conditions known to those skilled in the art to a polynucleotide sequence that encodes a protein that can direct cell migration or tissue shaping in the target organism. In one embodiment, a variant polynucleotide can hybridize under stringent hybridization conditions to a
C. elegans
gon-1 coding sequence. The variant polynucleotide sequence can be a polynucleotide obtained from an organism or can be a mutated version of any polynucleotide sequence noted above. The variant polynucleotide can encode a protein that is identical or altered relative to the wild-type
C. elegans
GON-1 protein. The encoded protein can have enhanced or reduced activity in vivo relative to GON-1.
In a related aspect, a polynucleotide coding sequence that encodes a protein having structural and functional similarity with a wild-type or altered migration or shaping protein can also be substituted, in whole or in part, with structurally related or unrelated sequences to encode a heterologous protein or a chimeric protein in the disclosed system, as detailed below.
Applicants herein disclose that the
Caenorhabditis elegans
gon-1 activity is encoded by a polynucleotide coding sequence (gon-1; SEQ ID NO:1) that encodes an essential protein (GON-1; SEQ ID NO:2) that directs migration of a growing gonadal tube through surrounding basement membranes during gonadogenesis in the nematode and also controls gonadal shape and organ localization.
The migration directing ability and tissue shaping ability are separable and depend upon whether the gon-1 coding sequence is expressed in distal tip cells or in muscle cells, respectively. In wild-type
C. elegans
, a gonad of normal shape is produced when gon-1 is expressed in both cell types. Accordingly, one aspect of the invention can also a method for shaping a tissue by selectively expressing a protein associated with both tissue e
Blelloch Robert H.
Kimble Judith E.
Quarles & Brady LLP
Shukla Ram R.
Wisconsin Alumni Research Foundation
LandOfFree
Agent and method for modulation of cell migration does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Agent and method for modulation of cell migration, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Agent and method for modulation of cell migration will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3202136