Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1997-03-19
2001-07-03
Elliott, George C. (Department: 1635)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S006120, C435S455000, C435S320100, C536S023100, C536S023500, C536S024100
Reexamination Certificate
active
06255071
ABSTRACT:
1. INTRODUCTION
The present invention relates to methods and compositions for the elucidation of mammalian gene function. Specifically, the present invention relates to methods and compositions for improved mammalian complementation screening, functional inactivation of specific essential or non-essential mammalian genes, identification of mammalian genes which are modulated in response to specific stimuli, identification of secreted proteins and cell packaging.
2. BACKGROUND
In yeast genetic systems, many options are available for delivery of gene sequences for the purpose of conferring a phenotype onto the host cell. For example, one common delivery system is a high copy plasmid system based on the endogenous yeast 2-micron plasmid. Plasmids from this origin achieve copy numbers of roughly 100 per cell and are randomly segregated to daughter cells upon division. In another system, the CEN system, CEN plasmids are maintained at low copy number (approximately 1 to 2 per cell) are segregated to daughter cells by the same mechanism used for segregation of the host chromosomes.
Further, methods have been devised in yeast by which the problems of gene isolation and discovery of gene function can be addressed efficiently. For example, in yeast it is possible to isolate genes via their ability to complement specific phenotypes. Further, in yeast, targeted insertional mutagenesis techniques can be used in yeast to inactivate or “knock out” a gene's activity. In mammalian systems, however, such methods are, in practical terms, lacking, which has made the elucidation of mammalian gene function a very difficult task.
For example, with respect to gene inactivation techniques in mammalian cells, the fact that mammalian cells are diploid and have complex genomes cause insertional mutagenesis techniques in mammalian systems to be a laborious, time-consuming and lengthy process.
Further, a major barrier to the development of such capabilities as complementation screening in mammalian cells has been that conventional techniques yield gene transfer efficiencies in most cells (0.01%-0.1%) that make screening of high complexity libraries impractical. While reports indicate that recombinant, replication deficient retroviruses can make possible increased gene transfer efficiencies in mammalian cells (Rayner & Gonda, 1994, Mol. Cell. Biol. 14:880-887; Whitehead et al., 1995, Mol. Cell. Biol. 15:704-710), retroviral-based functional mammalian cloning systems are inconvenient and have, thus far, failed to achieve widespread use.
The lack of convenience and impracticality of current retroviral-based cloning systems include, for example, the fact that the production of high complexity libraries has been limited by the low transfection efficiency of known retroviral packaging cell lines. Furthermore, no system has provided for routine, easy recovery of integrated retroviral proviruses from the genomes of positive clones. For example, in currently used systems the recovery of retrovirus inserts may be accomplished by polymerase chain reaction (PCR) techniques, however this is quite time consuming and variable for different inserts. Furthermore, with the use of PCR, additional cloning steps are still required to generate viral vectors for subsequent screening. Additionally, no mechanism has been available for distinguishing revertants from provirus-dependent rescues, a major source of false positives.
Further, it would be advantageous if an episomal system such as those found in yeast existed for efficient, broad spectrum use in mammalian systems. While bovine papillomaviruses (BPV), for example, replicate as extrachromosomal episomes, their use in developing episomal vectors has been limited.
Specifically, the ability of BPV replicate as episomes has been exploited in the past to create episomal vectors, using the so-called 69% fragment (T69). Vectors based upon T69 replicate in certain murine cell lines to give copy numbers that range from 15 to 500 copies per haploid genome, depending on the cell line. T69 vectors, however, exhibit a narrow host range. Further, the T69 fragment, like SV40, is oncogenic. Indeed, one method for identifying cells carrying T69 vectors specifically involves screening for transformed C127 cells.
3. SUMMARY OF THE INVENTION
The present invention relates to methods and compositions for the elucidation of mammalian gene function. Such methods can utilize novel integrating and/or episomal genetic delivery systems, thereby providing flexible, alternate genetic platforms for use in a wide spectrum of mammalian cells, including human cells. Specifically, the present invention relates to methods and compositions for improved mammalian complementation screening, functional inactivation of specific essential or non-essential mammalian genes, identification of mammalian genes which are modulated in response to specific stimuli, identification of mammalian genes that encode secreted products, and production and selection of novel retroviral packaging cell lines.
In particular, the compositions of the present invention include, but are not limited to, replication-deficient retroviral vectors, libraries comprising such vectors, retroviral particles produced by such vectors in conjunction with retroviral packaging cell lines, integrated provirus sequences derived from the retroviral particles of the invention and circularized provirus sequences which have been excised from the integrated provirus sequences of the invention.
The compositions of the present invention further include ones relating to improved mammalian episomal vectors. In particular, these compositions include, but are not limited to, expanded host range vectors (pEHRE), and libraries, cells and animals containing such vectors. The pEHRE vectors of the invention provide a consistent, stable, high-level episomal expression of gene sequences within a broad spectrum of mammalian cells. The pEHRE vectors of the invention comprise, first, replication cassettes in which papillomavirus (PV) E1 and E2 proteins are expressed from a constitutive transcriptional regulatory sequence or sequences, and, second, minimal cis-acting elements for replication and stable episomal maintenance.
The pEHRE vectors of the invention include, but are not limited to, vectors for delivery of sense and antisense expression cassettes, regulated expression cassettes, large chromosomal segments, and cDNA libraries, to a wide range of mammalian cells. Among the pEHRE vectors presented are ones which, additionally, can be utilized for the large scale production of recombinant proteins, and ones which can be utilized in the construction of cell lines that stably produce high titer viruses.
The compositions of the present invention further include novel viral packaging cell lines.
In particular, the methods of the present invention include, but are not limited to, methods for the identification and isolation of nucleic acid molecules based upon their ability to complement a mammalian cellular phenotype, antisense-based methods for the identification and isolation of nucleic acid sequences which inhibit the function of a mammalian gene, gene trapping methods for the identification and isolation of mammalian genes which are modulated in response to specific stimuli, methods for efficient large scale recombinant protein expression and methods for modulating the expression of known genes.
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Blasina, A. et al., “Copy-up mutants of the plasmid RK2 replication initiation protein are defective in coupling RK2 replication origins,”Proc. Natl. Acad Sci.USA (1996) 93:3559-3564.
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Beach David H.
Conklin Douglas
Hannon Gregory J.
Sun Peiqing
Cold Spring Harbor Laboratory
Elliott George C.
Foley Hoag & Eliot LLP
McGarry Sean
Olesen James T.
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