Functional characterization of genes

Details Functional characterization of genes Functional characterization of genes

1999-05-24

2001-10-09

Benzion, Gary (Department: 1638)

Multicellular living organisms and unmodified parts thereof and

Method of using a plant or plant part in a breeding process...

Method of breeding involving a genotypic or phenotypic marker

C800S278000

Reexamination Certificate

active

06300542

ABSTRACT:

BACKGROUND OF THE INVENTION
Modern advances in recombinant DNA technology have made possible the cloning and sequence determination of many genes, the functions of which remain to be determined. Methods have been developed for ascertaining the functions of these genes and may be divided into three main types.
The first method involves the sequestration of the messenger RNA corresponding to the gene sequence of interest by an antisense oligonucleotide or nucleotide analogue complementary to the sequence of the RNA. This prevents initiation of translation of the messenger RNA by the ribosome, and production of the protein product of the gene is thereby suppressed. The function of the gene is therefore imputed from observation of a mutant phenotype induced by this suppression. Introduction of the antisense oligonucleotide may be achieved either by transfection of the target cell with a suitable gene construct which directs production of an antisense transcript within the cell, or by direct introduction of exogenous oligonucleotides or oligonucleotide analogues into the cell.
Although the antisense method has been successful in model systems such as cultures of dissociated cells, its implementation in a whole organism is problematic. For example, simultaneous delivery of exogenous oligonucleotides to all tissues of interest in a whole organism is extremely difficult. Exogenous oligonucleotides are also metabolized by cells, and any phenotype produce by this method is therefore only temporary. To preserve a desired mutant phenotype for prolonged study the mutation must be made heritable by creation of a transgenic organism. This requires that a gene construct coding for the antisense RNA be stably creation of a transgenic organism. This requires that a gene construct coding for the antisense RNA be stably transfected into the germ cells of the organism under study, followed by rounds of inbreeding of transgenic progeny to make organisms homozygous for the mutation. Creation of transgenic organisms is therefore typically a laborious and expensive process.
A second approach to determine the function of a gene of known sequence is by the “co-suppression” technique, which also requires the creation of a transgenic organism. In this approach the transfected gene construct codes for the gene of interest in the sense orientation, and a small proportion of the transformants exhibit loss of function of the gene of interest via an unknown trans mechanism. As with the antisense approach this method suffers from the disadvantage of requiring the creation of a transgenic organism.
A third approach is to disrupt the gene of interest by transformation of target cells with a vector designed to stably integrate into the host chromosome within the coding sequence of the gene of interest, via the process of homologous recombination. Since homologous recombination is a rare event, this approach typically utilizes a targeting vector designed to introduce a gene for antibiotic resistance normally lacking in the target cells, allowing selection of the small number of desired transformants. Integration of the antibiotic resistance gene within the coding sequence of the gene of interest in this fashion also serves to disrupt normal transcription of the gene, producing an aberrant, non-functional protein product. This approach also requires germ-line transmission of the disrupted gene for subsequent generation of organisms homozygous for the mutation, and therefore also suffers from the disadvantages discussed supra.
A more recent method to cause gene disruption has been the use of naturally occurring transposable elements to introduce gene insertions, together with the use of a PCR to detect an insertion event within a particular gene of interest. Ballinger et al.,
Proc. Nat'l Acad. Sci.
86: 9402-06 (1989); Kaiser et al., loc. cit. 87: 1686-90 (1990); Zwaal et al., loc. cit. 90: 7431-35 (1993). In this method two oligonucleotide primers were employed in the PCR, with one primer complementary to a sequence within a particular gene of interest, and the other primer complementary to a portion of the tandem inverted repeat sequence of the transposable element.
Both Ballinger et al. and Kaiser et al. crossed parental strains of the fruit fly
Drosophila melanogaster,
one strain of which carried a transposable element, to produce a pool of heterozygous F
1
progeny bearing genome insertions caused by the transposable element. Ballinger et al. then screened the genomic DNA of the F
1
progeny for the presence of insertions in two genes of unknown function, as described above. Flies in which the desired gene insertions were observed were then used to produce F
2
progeny homozygous for the insertion, and their phenotype examined for effects attributable to the absence of function of the gene of interest. But no phenotypic changes from the wild type were observed for any F
2
flies which were homozygous for insertions into either gene of interest.
Kaiser et al. carried out the screening for gene insertions using the DNA of the segregating F
2
generation of flies, rather than at the F
1
generation. Insertions were observed into a gene which was previously known to produce a specific phenotype when disrupted by insertion, and the expected phenotype was observed. In this fashion Kaiser et al. demonstrated that disruption of a known gene by insertion of a transposable element could be correlated with observation of a previously known phenotype.
An additional problem associated with DNA analysis of the F
2
generation instead of the F
1
generation is the possibility of observing additional insertion events into the gene of interest caused by the activity of the transposable element during generation of the F
2
generation. These mutant alleles will be detected by DNA analysis of the F
2
generation, but as they will not genetically segregate until the next gametic cycle they will not contribute to the homozygous genotype of the F
2
generation. Accordingly, DNA sampling at the F
2
generation will lead to the identification by PCR of insertion events in the gene of interest which will not be reflected in the phenotype of the F
2
generation, generating false positive results which require further experimentation to detect.
Zwaal et al., working in the worm
Caenorhabditis elegans
(
C. elegans
), also carried out PCR analysis for gene insertion events caused by a transposable element in the DNA of the F
2
and subsequent generations. In addition the Tc1 transposable element was used which also generated deletion mutants in the F
2
and subsequent generations caused by the transposable element “jumping” from the initial site of insertion and excising some amount of DNA from the region of the gene flanking the insertion site. Despite the detection of 23 insertion events into 16 known genes and 7 deletion events in 6 known genes, no phenotypic difference from the wild type was observed in any of the F
2
worms or subsequent generations.
Zwaal et al. also described the production of libraries of F
2
progeny of
C. elegans
in a frozen state. These libraries could be used both to prepare DNA for PCR analysis as described above, and to recover viable worms for subsequent phenotypic analysis. Use of such libraries could in principle obviate the need to generate new collections of mutant progeny in order to analyze the function of each new gene of interest. The libraries disclosed, however, allow the preparation of only small quantities of DNA which may be sampled only a limited number of times, and which are insufficient for distribution to other laboratories. In order to detect insertions in more than a small number of genes, therefore, frequent generation of new libraries will still be required.
Although the aforementioned techniques are available for determining the function of a gene of known sequence, each conventional methodology has significant drawbacks, and the development of a rapid, inexpensive method would be highly desirable.
SUMMARY OF THE INVENTION
It is therefore an object of the present invention

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