Expression of DNA or proteins in C. elegans

Details Expression of DNA or proteins in C. elegans Expression of DNA or proteins in C. elegans

2000-03-16

2002-10-15

Crouch, Deborah (Department: 1632)

Multicellular living organisms and unmodified parts thereof and

Nonhuman animal

Transgenic nonhuman animal

C800S003000

Reexamination Certificate

active

06465715

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to the expression of DNA, genes, cDNAs, proteins, peptides and parts thereof in the excretory canal of the nematode worm
C. elegans.
In particular, the invention relates to promoter sequences which are capable of directing tissue-specific gene expression in the excretory canal of
C. elegans,
to expression vectors containing the promoter sequences, to transgenic
C. elegans
specifically expressing reporter genes in the excretory canal, to methods of identifying chemical agents that affect the morphology of the excretory canal and to use of these agents in the pharmacological treatment of diseases for which the
C. elegans
excretory canal serves as a model.
BACKGROUND OF THE INVENTION
The
C. elegans
Excretory Cell
The excretory system of the nematode
C. elegans
consists of three cells: a single large excretory cell, a duct cell and a pore cell that interfaces with the duct to the main body hypodermis. The excretory cell is the largest mononucleate cell in
C. elegans.
The nucleus and cell body of the excretory cell is situated at the terminal bulb of the pharynx. The cell itself is shaped in an H-form, with the two arms situated along the lateral lines for almost the entire length of the worm, and slightly dorsal. The excretory cell is polarized, having an apical domain facing the lumen of the excretory canal and a basal domain facing outside. The structure and the organization of the
C. elegans
excretory system suggest that it may be used for osmoregulation and can therefore be considered as a model for the vertebrate nephron.
Various mutant
C. elegans
have been reported which have an aberrant phenotype in the excretory canal. These aberrant phenotypes include cyst formation, short canals and branched canals. Various mutations affecting the excretory canal can be traced back in
C. elegans
II, ed. Riddle, Blumenthal, Meyer and Priess, Cold Spring Harbor Laboratory Press, 1997.
Drug Discovery in Growth Cone Steering.
Regulation of cell motility, cell shape and the outgrowth of axons or other cell outgrowths are all essential processes in the morphogenesis and function of both unicellular and multicellular organisms. Furthermore, the control of these processes is disturbed in a variety of diseases in which receptors, extra-cellular signals and intra-cellular pathways are over- or under-stimulated. The discovery of new genes, proteins and peptides that are involved in these processes and chemical entities which modulate them would very much help the understanding of these processes. Accordingly, there is a need to develop new methods for the discovery of novel molecules involved in the cell motility, cell shape and cell outgrowth process, and to establish their function. In addition, since malfunction of these biological processes can lead to disease there is also a need to discover chemical entities which modulate these processes which may be useful as pharmaceuticals. Diseases associated with cell motility, cell shape and cell outgrowth include cancerous disease, more particularly tumor formation, tumor metastasis and vascularisation of tumors.
Drug Discovery in Renal Diseases.
In the drug discovery process it is established practice to develop a model of a disease which can be used in the development of assays to screen for compounds with potential pharmaceutical activity. For kidney diseases, and more specifically kidney cyst formation, two different types of disease models currently exist; models based on cell cultures of renal epithelial cells and mouse models. Although these systems have been presented as models for cystic diseases, such as autosomal dominant polycystic kidney disease (ADPKD), they have several disadvantages.
The models based on cell cultures can never be compared with a live multicellular organism. Where aberrant growth indicative of cyst formation has been observed in cultures of different cells, it has proven difficult to develop efficient compound screens from these models. Furthermore, even if chemicals can be discovered that modulate cell growth and hence cyst formation in culture, it remains difficult to prove that these compounds will have analogous effects in the renal systems of multicellular organisms.
The developed mouse models for renal cyst diseases have the disadvantage that they are not suitable for middle to high throughput screening for the discovery of pharmacological compounds. Accordingly, there remains a clear need for an alternative model of renal diseases which more accurately models the renal systems of multicellular organisms but which is practical for use in middle to high throughput screening.
SUMMARY OF THE INVENTION
The present invention relates to the use of the
C. elegans
excretory cell in the drug discovery process. The
C. elegans
excretory canal is an efficient tool to study various developmental biological features; it is formed during the larval stages of the nematode and the canals are observed to grow along the animal in early development. Hence, the development of the excretory canal is an efficient tool to study growth cone steering and defects that might arise during its development and the excretory canal can be used as a model for the development of drug screens in the area of growth cone steering and directional outgrowth.
The
C. elegans
excretory cell and excretory canal can also be considered as a model of the human kidney nephron. The excretory canal has analogous apical-basal polarities as can be found in certain kidney cells and which are relevant for cellular function. Hence, studying the excretory canal may help to develop new tools against kidney diseases. Furthermore, the excretory canal can be used as a model for the development of drug screens in the area of kidney diseases.
In order to exploit the potential of the
C. elegans
excretory cell and excretory canal both as a disease model and in the development of drug screens it would be advantageous to be able to express any gene or cDNA of interest, including reporter genes, specifically in the excretory cell and excretory canal. To achieve this would require the identification of a tissue-specific promoter which is active in the excretory cell.
The present inventors have identified, through the use of biochemical, molecular biology and transgenic techniques, a promoter fragment that specifically directs transcription in the
C. elegans
excretory cell in a very efficient way. From this promoter fragment several deletions have been generated that still promote transcription, and hence gene expression, in the excretory cell of
C. elegans.
These promoter fragments are useful tools as they can be used to direct specific expression of any DNA fragment of interest in the excretory cell and excretory canal.
Accordingly, in a first aspect the invention provides a DNA fragment which is capable of functioning as a promoter directing gene expression in the excretory cell of
C. elegans,
which DNA fragment comprises the sequence of nucleotides set forth in any one of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or a fragment thereof in the absence of any other sequence of consecutive nucleotides from the
C. elegans
genome (i.e., an isolated DNA fragment).
According to another aspect of the invention, an isolated nucleic acid molecule, is provided. The isolated nucleic acid molecule can comprise: (a) nucleic acid molecules which hybridize under stringent conditions to a molecule consisting of a nucleic acid having a sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7, and which direct expression of a heterologous DNA fragment to the excretory canal of
C. elegans,
(b) deletions, additions and substitutions of (a) which direct expression of a heterologous nucleic acid to the excretory canal of
C. elegans,
and (c) complements of (a) or (b) which direct expression of a heterologous nucleic acid to the excretory canal of
C. elegans.
According to another aspect of the invention,

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