Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-09-11
2004-03-09
Ketter, James (Department: 1636)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S484000
Reexamination Certificate
active
06703200
ABSTRACT:
BACKGROUND
The present invention relates to methods and materials for the systematic and random insertion of genetic material into the genome of an organism. The invention allows the rapid mutagenesis of organisms to mutate essentially every gene of an organism, particularly fungi, and allow the reliable and efficient identification of the gene being knocked out in each mutagenesis event. The invention also facilitates very high efficiency of homologous recombination, particularly in species, such as filamentous fungi, that have previously been notorious for low frequency of such events.
Numerous methods for introducing foreign genetic material into living cells have become routine since the first instances of genetic engineering almost a quarter century ago. Introduction of foreign genetic material can be into the cell via a vector that may replicate or by incorporation into the genome of the host cell. The introduction of such foreign genetic material has allowed the expression of a protein in a species that usually does not produce the protein. It has also allowed the regulation of the expression of a protein (overexpression and underexpression) by introducing modified regulatory sequences making the transcription and translation of the protein more or less efficient. Another use for genetic engineering has been the modification of the biological activity of a structural protein or enzyme by altering the coding region of a gene and thus altering the amino acid sequence of the protein produced. The altered amino acid sequence can lead to changes in conformation, changes in surface charge, and changes in the higher structure of the protein (tertiary and quanternary structure) which all can lead to changes in biological activity.
With the recent growth of the field of “functional genomics” out of the discipline of genomics or gene sequencing, the manipulation of DNA in organisms has taken on another urgent task. In addition to sequencing the genetic material of an organism, functional genomics seeks to identify the function of the genes of a target organism on an industrial scale. By determining the function of most, if not all, genes and the products of those genes in an organism, functional genomics can accelerate the identification of gene and protein targets and allow the identification of compounds that will modulate those genes and gene products to alleviate disease, improve human and animal health, and improve the quality and quantity of food crops. To achieve this, it is necessary to develop rapid, high volume techniques for sytematically altering the expression of essentially every gene in an organism, identifying the corresponding gene and monitoring the effect of the gene alteration on the phenotype of the organism.
Automated processes in molecular genetics have allowed the systematic analysis of genomes from microorganisms, such as yeast and bacteria, by DNA sequencing. Attention is focused on rapidly ascribing functions to newly discovered genes. It is widely recognized in the field of genetics that gene function is most desirably assigned through the analysis of organisms containing defined gene mutations (mutants).
Previous methods of introducing genetic material into a eukaryotic organism are sufficient for mutating a single gene. Such methods include protoplast fusion, transformation by electroporation, particle bombardment, chemical perturbation of cellular envelopes (membranes and walls), phage and viral infection, transduction and physical insertion of DNA into cells. Many of these methods are limited to introducing DNA into a cell in the form of a vector, where the DNA is expressed to produce its gene product. The desired characteristics of a useful gene insertion method for functional genomics include the insertion of a gene or DNA fragment into essentially every gene in the genome of the target organism in an efficient and systematic manner. Methods for getting DNA to integrate into the genome of an organism usually insert the DNA randomly into the genome. Methods for getting DNA to integrate into a specific location in the genome of an organism are less reliable and often have low efficiency.
Random insertion of DNA into another piece of DNA, including genomic DNA, include viral systems that use recombinases such as Cre-lox (Sauer (1996) Nucleic Acid Res. 24:4608-4613) and Flp recombinase (Seibler and Bode (1997) Biochemistry 36:1740-1747). These systems insert DNA at specific sites in DNA in genomic DNA of a host, but the specific sites have been randomly engineered into the genome. Recently, the ability of enzymes known as transposases to transfer DNA fragments from one location in DNA into another random location in DNA have been discovered (Devine et al., U.S. Pat. No. 5,677,170; Devine et al., U.S. Pat. No. 5,728,551; Hackett et al., WO 98/40510; Plasternak et al., WO 97/29202; Reznikoff et al., WO 98/10077; Craig WO 98/37205; Strathman et al., (1991) Proc. Nat. Acad. Sci. USA 88:1247-1250; Phadnis et al., (1989) Proc. Nat. Acad. Sci. USA 86:5908-5912; Way et al., (1984) Gene 32:269-279; Kleckner et al., (1991) Method. Enzymol. 204:139-180; Lee et al., (1987) Proc. Nat. Acad. Sci. USA 84:7876; Brown et al. (1987) Cell 49:347-356; Eichinger et al. (1988) Cell 54:955-966; Eichinger et al. (1990) Genes Dev. 4:324-330). Generally, the transposase recognizes a relatively short DNA sequence known as an inverted repeat that is located on the flanks of an internal piece of DNA. The DNA sequence comprising the internal DNA sequence and the two flanking internal repeat sequences is known as a transposon or transposable element. The transposase has the ability to excise the transposon and insert it in another piece of DNA into which it comes into contact. The location of the insertion is usually not totally random and occurs preferentially at target sequence locations (so called “hot spots”)(Kleckner et al., (1991) Method. Enzymol. 204:139-180). Like the viral systems, the insertions are site specific, but the sites are randomly located in the genome and do not allow site directed insertion.
Transposons have been used to introduce a desired gene into an organism, wherein the introduction is via a plasmid or randomly (site specific, but not site directed) into the organism's genome. Another use of transposons is as a sequencing tool since the sequence of the transposon is often known, especially at the borders, such that use of primers designed for the transposon would allow sequencing of the DNA into which the transposon is inserted. The lack of randomness in insertion location would detract from the use of transposons as tools to systematically sequence essentially all genes in an organism or to systematically knock out essentially all genes in an organism. Therefore, their use in functional genomics would appear to be limited.
Using transposons has thus far involved engineering the transposon into a plasmid (e.g., Reznikoff et al., WO 98/10077) and introducing the plasmid into a target organism such that the transposed gene is expressed by the plasmid (Devine et al., U.S. Pat. No. 5,677,170; Devine et al., U.S. Pat. No. 5,728,551). Alternatively, genetic material has been introduced into the genome of an organism by directly transferring the transposon from a plasmid to the genome of a target organism in the presence within the cell of the transferring transposase (Hackett et al., WO 98/40510; Plasternak et al., WO 97/29202). For this to occur, the interior of the cell to be transposed must include a transposable element on a plasmid and the corresponding transposase. Consequently, the only use of transposons to get DNA into the genome of an organism using a transposon has been to directly transpose the transposable DNA in the presence of a transposase into a site specific, but not site directed location (Hackett et al., WO 98/40510; Plasternak et al., WO 97/29202). Additionally, vectors containing a transposon event have been limited to plasmids and the use of the transposed vectors has been the expression of the transposed gene's gene product.
Hamer John E.
Hamer Lisbeth
Hofmeyer Timothy G.
Ketter James
Kiefer Laura L.
Paradigm Genetics, Inc.
Spencer Deborah H.
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