Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1997-09-29
2002-03-12
Carlson, Karen Cochrane (Department: 1653)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
Reexamination Certificate
active
06355415
ABSTRACT:
FIELD OF THE INVENTION
The present invention provides nucleic acid sequences and methods to determine gene function. In particular, the present invention relates to reducing the levels of RNA encoded by a DNA sequence of interest using ribozymes. The present invention further relates to ribozyme sequences, recombinant expression vectors encoding ribozymes as well as host cells and transgenic animals comprising such expression vectors.
BACKGROUND OF THE INVENTION
The human genome project and allied interests will soon have elucidated the sequence of the entire human genome [Cox et al (1994)
Science
265:2031-2031; Guyer et al (1995)
Proc. Natl. Acad. Sci. USA
92:10841-10848]. While this anticipated advance is exciting, it is also misleading since knowledge of the sequences of open reading frames and genetic coding regions, without a knowledge of the function of the gene products of this vast array of putative genes, provides only very limited insight into the human genome. Full knowledge of the genome requires knowledge of the function of each of the gene products of the putative genetic coding sequences. While gene function determination is ongoing within the field of molecular genetics, the rate at which the function of a gene can be determined is many orders of magnitude slower than the rate at which a gene can be sequenced. Therefore, a massive backlog of genetic sequences in search of a function looms on the horizon.
One of the traditionally used means of determining gene function is by “knocking out” or disrupting the coding sequence in an animal model and observing which structure(s) or function(s) is deleted in the resulting “knock out” model [Capecchi et al (1989)
Science
244:1288-1292; Hasty et al (1991)
Nature
350:243-246]. Gene knockouts are presently accomplished by homologous recombination in embryonic stem (ES) cells with a targeting vector, production of mosaic animals (in particular, mice) with a single disrupted allele in some of their germ cells, breeding to mice which are heterozygous for the disrupted gene, and finally inbreeding to venerate homozygous mice where the targeted gene disruption is present in each allele so that the full gene complement is rendered non-functional [Shastry et al (1994)
Mol. Cellul. Biochem
. 136:171-182; Galli-Taliadoros et al (1995)
J. Immunol. Methods
181:1-15]. Only when each of these very time consuming steps has been successfully accomplished will a gene knockout mouse, which will show the phenotype associated with deletion of the targeted gene, be available for further determination of the function of the deleted gene. Clearly, this is a complex and slow method for gene function determination and cannot be expected, in a reasonable time, to yield the function of the many tens of thousands of coding sequences elucidated by the present flurry of genetic sequencing.
Therefore, there remains a need for a rapid and specific means for the determination of gene function.
SUMMARY OF THE INVENTION
The present invention provides methods for the identification of one or more functions of a nucleotide sequence in an organism which are useful for the rapid identification of, for example, disease related genes which may be targeted for the treatment or prevention of disease. The invention further provides recombinant expression vectors which encode ribozyme sequences, transgenic host cells, transgenic embryos and transgenic organisms which express a ribozyme sequence that is capable of cleaving mRNA encoded by the nucleotide sequence whose function is sought to be determined. The ribozyme sequences, recombinant expression vectors, transgenic host cells, transgenic embryos and transgenic organisms provided by the present invention are useful in identifying the function of any sequence of interest in an organism.
In one embodiment, the invention provides a ribozyme sequence capable of cleaving RNA, wherein the RNA is encoded by SEQ ID NO:5. While not restricted to any particular ribozyme sequence, in one preferred embodiment, the ribozyme sequence is selected from the group consisting of SEQ ID NO:19, SEQ ID NO:20 and SEQ ID NO:21.
In another embodiment, the invention contemplates a DNA sequence encoding a ribozyme sequence capable of cleaving RNA encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NO:5 and SEQ ID NO:27. Although it is not intended that the DNA sequence be limited to any particular sequence, in one preferred embodiment, the DNA sequence is selected from the group consisting of DNA sequences encoding SEQ ID NO:19, SEQ ID NO:20, and SEQ ID NO:21.
In yet another embodiment, the invention provides a recombinant expression vector comprising a DNA sequence encoding a ribozyme sequence capable of cleaving RNA encoded by SEQ ID NO:5. While not restricted to any ribozyme sequence, in a preferred embodiment, the ribozyme sequence is selected from the group consisting of SEQ ID NO:19, SEQ ID NO:20 and SEQ ID NO:21. In an alternative embodiment, it is preferred, though not required, that the recombinant expression vector is selected from the group consisting of pT
7
vaRz
435
, pT
7
vaRz
365
, and pT
7
vaRz
564
. In a particularly preferred embodiment, the recombinant expression vector is selected from the group consisting of pT
7
GaRz
435
, pT
7
GaRz
365
and pT
7
GaRz
564
.
Also provided by the invention is a host cell comprising a recombinant expression vector wherein the recombinant expression vector comprises a DNA sequence encoding a ribozyme sequence capable of cleaving RNA encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NO:5 and SEQ ID NO:27. While not restricted to a particular host cell, one preferred embodiment contemplates that the host cell is a fertilized egg.
The invention further provides a transgenic zebrafish cell comprising a heterologous ribozyme sequence capable of cleaving an RNA sequence comprised in the zebrafish cell. In one preferred embodiment, the transgenic zebrafish cell is a fertilized egg. In an alternative embodiment, the transgenic zebrafish cell is an embryonic cell. In yet another alternative embodiment, the transgenic zebrafish cell is an adult cell.
The present invention further provides a method for identifying one or more functions of a DNA sequence of interest comprised in a genome of an organism, comprising: a) providing: i) a cell derived from the organism, wherein the cell comprises the DNA sequence of interest encoding RNA sequence; and ii) a ribozyme sequence capable of cleaving the RNA sequence; b) introducing the ribozyme sequence into the derived cell to generate a manipulated cell, wherein the introducing is under conditions such that the RNA sequence is cleaved by the ribozyme sequence; and c) detecting one or more changes in the manipulated cell relative to the derived cell thereby identifying one or more functions of the DNA sequence in the organism.
The present invention also provides a method for identifying one or more functions of a DNA sequence of interest comprised in a genome of an organism, comprising: a) providing: i) a cell derived from the organism, wherein the cell comprises the DNA sequence of interest encoding RNA sequence; and ii) a ribozyme sequence capable of cleaving the RNA sequence; b) introducing the ribozyme sequence into the derived cell to generate a manipulated cell, wherein the introducing is under conditions such that the RNA sequence is cleaved by the ribozyme sequence; c) permitting the manipulated cell to generate progeny cells; and d) detecting one or more changes in at least one of the progeny cells relative to the derived cell thereby identifying one or more functions of the DNA sequence of interest in the organism.
Also provided by the present invention is a method for identifying one or more functions of a first DNA sequence of interest comprised in a genome of a first organism, comprising: a) providing: i) a cell derived from a second organism, wherein the derived cell comprises a genome comprising a second DNA sequence homologous to the first DNA
Wagner Thomas E.
Xie Yuefeng
Carlson Karen Cochrane
Medlen & Carroll LLP
Ohio University
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