Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
1999-04-19
2004-08-17
Nguyen, Dave T. (Department: 1635)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
C435S006120, C435S325000, C435S320100, C435S465000, C435S462000, C435S463000, C536S023200, C536S023500, C536S023700, C800S018000
Reexamination Certificate
active
06777235
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to gene entrapment vectors and their use in gene discovery, and their use in screening for or making cells and organisms that are mutated for such genes. This invention also relates to the use of such entrapment vectors to identify tissue specific transcription control elements such as promoters and enhancers and for generating transgenic animals displaying restricted expression of transgenes. This invention also relates to trap vectors comprising a splice acceptor and a sequence encoding a reporter gene.
BACKGROUND OF THE INVENTION
Genomic based drug discovery is largely dependent upon the identification of specific genomic targets. Thus, cloning, sequencing, and identification of function of mammalian genes is a first priority in a genomic based drug discovery. In particular, it is important to identify and make use of genes which are spacially and/or temporally regulated in the organism.
Animal model systems such as the fruit fly and the worm are often used in gene identification because of ease of manipulation of the genome and ability screen for mutants. While these systems have their limitations, large numbers of developmental mutations have been identified in those organisms either by monitoring the phenotypic effects of mutations or by screening for expression of reporter genes incorporated into developmentally regulated genes.
Many features of the mouse make it the best animal model system to study gene function. However, the mouse has not been used for large scale classical genetic mutational analysis because random mutational screening and analysis is very cumbersome and expensive due to long generation times and maintenance costs.
A disadvantage in using animal models for the identification of genes is the need to establish a transgenic animal line for each mutational event. This disadvantage is alleviated in part by using embryonic stem (ES) cell lines because mutational events may be screened in vitro prior to generating an animal. ES cells are totipotent cells isolated from the inner cell mass of the blastocyst. Methods are well known for obtaining ES cells, incorporating genetic material into ES cells, and promotion of differentiation of ES cells. ES cells may be caused to differentiate in vitro or the cells may be incorporated into a developing blastocyst in which the ES cells will contribute to all differentiated tissues of the resulting animal. Vectors for transforming ES cells and suitable genes for use as reporters and selectors are also well known.
Gene entrapment strategies have been employed to identify developmentally regulated genes. One type of entrapment vector is called a “promoter trap”, which consists of a reporter gene sequence lacking a promoter. Its integration is detected when the reporter is integrated “in-frame” into an exon. “Gene trap vectors” target the much more prevalent introns of the eucaryotic genome. The latter vectors consist of a splice-acceptor site upstream from a reporter gene. Integration of the reporter into an intron results in a fusion transcript containing RNA from the endogenous gene and from the reporter gene sequence.
Gene trap vectors may be made more efficient by incorporation of an internal ribosomal entry site (IRES) such as that derived from the 5′ non-translated region of encephalomyocarditis virus (EMCV). Placement of a IRES site between the splice acceptor and the reporter gene of a gene trap vector means that reporter gene product need not be translated as a fusion product with the endogenous gene product, thereby increasing the likelihood that integration of the vector will result in expression of the reporter gene product.
Examples from the literature of the use of promoter and gene trap vectors as well as such vectors including an IRES sequence, are listed below. Some examples involve the identification of developmentally regulated or tissue specific events making use of ES cell lines.
1. Canadian Patent application no. 2,166,850 (open for public inspection Jul. 11, 1996)
Vectors and the Use Thereof for Capturing Target Genes
: describes the use of transmembrane sequence encoding gene trap vectors to isolate and identify secretory proteins. Also see U.S. Pat. No. 5,767,336 issued Jun. 16, 1998.
2. U.S. Pat. No. 5,364,783 issued Nov. 15, 1994
. Retrovirus Promoter Trap Vectors
: describes retroviral vectors that are used to isolate transcriptionally active chromosomal regions and to identify promoter sequences. The reporter gene is placed in the U3 or U5 control region of the retrovirus.
2. Gossler, A., et al. (1989).
Mouse Embryonic Stem Cells and Reporter Constructs to Detect Developmentally Regulated Genes
. Science 244:463-465: describes the use of enhancer trap gene trap vectors for use in identifying developmentally regulated genes. The gene trap vector consists of the mouse En-2 splice acceptor upstream from lacZ (reporter) and a selector gene (hBa-neo).
3. Von Melchner, H., et al.
Isolation of Cellular Promoters by Using a Retrovirus Promoter Trap
. Proc. Natl. Acad. Sci. USA 1990, 87:3733-3737.
3. MacLeod, D., et al.:
A Promoter Trap in Embryonic Stem (ES Cells Selects for Integration of DNA into CpG Islands
. Nucleic Acids Res. 1991, 19:17-23.
4. Reddy, S., et al.:
Retrovirus Promoter-Trap Vector to Induce lacA Gene Fusions in Mammalian Cells
. J. Virol. 1991, 65:1507-1515.
5. Brenner, D. G., et al.:
Analysis of Mammalian Cell Genetic Regulation in Situ by Using Retrovirus-Derived Portable Exons Carrying the Escherichia coli lacZ Gene
. Proc. Natl. Acad. Sci. USA. 1989, 86:5517-5521.
6. Kerr, W. G., et al.:
Transcriptional Defective Retroviruses Containing lacZ for the in Situ Detection of Endogenous Genes and Developmentally Regulated Chromatin
. Cold. Spring. Harb. Symp. Quant. Biol. 1989, 54:767-776.
7. Friedrich, G. and Soriano, P.:
Promoter Traps in Embryonic Stem Cells: A Genetic Screen to Identify and Mutate Developmental Genes in Mice
. Genes. Dev. 1991, 5:1513-1523.
8. Skarnes, W. C., et al.:
A Gene Trap Approach in Mouse Embryonic Stem Cells: The lacZ Reporter is Activated by Splicing, Reflects Endogenous Gene Expression, and is Mutagenic in Mice
. Genes Dev, 1992, 6:903-918: describes gene trapping that results in activation of lacZ by splicing to endogenous exons and production of a fusion protein whose expression pattern mimics that of the endogenous gene. The resulting integration and fusion is mutagenic. Using 5′ RACE, the endogenous gene activated with three lacZ-based gene-trap insertions was cloned and the effectiveness of the En-2 splice acceptor site was demonstrated. For two insertions, the pattern of lacZ expression in embryos was shown to match the normal distribution of endogenous transcripts. Two of the three insertions tested cause phenotypic abnormalities in mice. One of those was an insertion into a novel gene expressed widely during development that causes perinatal death in homozygous animals. The other is an insertion into a zinc-finger gene expressed in neural cells that results in mild growth retardation after birth.
9. Von Melchner, H., et al.:
Selective Disruption of Genes Expressed in Totipotent Embryonal Stem Cells
. Genes. Dev. 1992, 6:919-927: where sequences upstream of nine retroviral promoter-trap insertions were cloned using if inverse PCR. Flanking probes from five ES cell lines detected transcripts, and one clone is identified as the REX-I transcription factor. Two of four lines transmitted to the germline caused embryonic-lethal phenotypes.
10. Sheriden, U., et al.:
Transcriptionally Active Genomic Regions are Preferred Targets for Retroviral Integration
. Mol. Cell. Biol. 1990, 64:907-912.
11. Vijaya, S., et al.:
Acceptor Sites for Retroviral Integrations Map Near DNase
1-
Hypersensitive Sites in chromatin
. J. Virol. 1986, 60:683-692.
12. Rohdewold, H., et al.:
Retrovirus Integration and Chromatin Structure: Moloney Murine Leukemia Proviral Integration Sites Map near DNAse I Hypersensitive Sites. J. Virol
1987, 61:336-343.
13. Boggs, S. S., et al.:
Efficient Transformati
Jirik Frank R.
Ong Christopher J.
Priatel John J.
Nguyen Dave T.
Schnizer Richard
The University of British Columbia
Townsend & Townsend & Crew LLP
LandOfFree
Complementation trap does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Complementation trap, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Complementation trap will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3349311