Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
2002-01-07
2003-11-11
Ketter, James (Department: 1636)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
C435S477000, C435S483000, C435S320100, C435S254210
Reexamination Certificate
active
06645766
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to novel shuttle vectors, and more particularly, shuttle vectors capable of replication in at least yeast and capable of expression in at least a mammalian cell.
BACKGROUND OF THE INVENTION
The introduction of cloned nucleotide sequences into mammalian cells has greatly facilitated the study of the control and function of various eukaryotic genes. Mammalian cells provide an environment conducive to appropriate protein folding, post translational processing, feedback control, protein-protein interactions, and other eukaryotic protein modifications such as glycosylation and oligomerization. Thus, a number of expression vectors have been developed which allow the expression of a polypeptide in a mammalian cell.
The typical mammalian expression vector will contain (1) regulatory elements, usually in the form of a viral promoter or enhancer sequences; (2) a multicloning site, usually having specific enzyme restriction sites to facilitate the insertion of a DNA fragment with the vector; and (3) sequences responsible for intron splicing and polyadenylation of mRNA transcripts. Generally, sequences facilitating the replication of the vector in both bacterial and mammalian hosts and a selection marker gene which allows selection of transformants in bacteria are also included. The bacterial elements, or in some cases phage elements, are included to provide the option of further analyzation of the nucleic acid inserts amplified and isolated from the bacteria or phage.
In the past, the insertion of a heterologous nucleic acid (insert) into the multicloning site of a mammalian expression vector has generally been accomplished by one of two methods. In the first method, the insert is cut out of a bacterial expression vector and ligated into the mammalian expression vector. In the second method, often called “TA cloning”, special ends are generated on the insert by PCR such that the modified insert can be put into the mammalian expression vector. Each of these methods requires a number of steps including enzymatic reactions which can be labor intensive and unreliable. Moreover, cloning efficiency drops significantly as the size of the insert increases.
Another method used for inserting a heterologous nucleic acid (insert) into an expression vector takes advantage of yeast's high efficiency at homologous recombination in vivo. In this method, a nucleic acid fragment flanked by 5′ and 3′ homologous regions is co-introduced into a yeast with a vector which has regions identical to the 5′ and 3′ regions flanking the fragment. The yeast efficiently homologously recombines such that the fragment inserts into the region of the vector flanked by the before-mentioned 5′ and 3′ regions. H. a., et al.,
Plasmid,
38:91-96 (1997), incorporated herein. Unfortunately, yeast are the only organisms able to efficiently recombine so as to insert heterologous nucleic acids into a vector. Therefore, to date, there is not an efficient method or means to transfer inserts into a specific region of a vector used for expression in mammalian cells.
Accordingly, it is an object of the invention to provide compositions and methods useful in facilitating the insertion of a heterologous nucleic acid into a vector which can express the heterologous nucleic acid in at least a mammalian cell.
Moreover, it is the object of this invention to provide a shuttle vector and methods of use which allow replication of the shuttle vector at least in yeast and which allow expression in at least a mammalian cell.
SUMMARY OF THE INVENTION
The invention provides shuttle vectors, and methods of using shuttle vectors, capable of expression in at least a mammalian cell. Furthermore, the shuttle vectors are capable of replication in at least yeast, and optionally, bacterial cells.
In one aspect of the invention, the invention provides a shuttle vector comprising an origin of replication functional in yeast and preferably, a reporter gene functional in yeast. The shuttle vector further comprises a promoter functional in a mammalian cell, capable of directing transcription of a polypeptide coding sequence operably linked to said promoter.
In another aspect of the invention, the shuttle vector comprises an insertion site operably linked to said promoter. The insertion site preferably allows for homologous recombination with a heterologous nucleic acid. In one embodiment, the insertion site has 5′ and 3′ regions identical to 5′ and 3′ regions flanking a nucleic acid to be inserted into the vector.
Optionally, the shuttle vector comprises any one or more of the following: an internal ribosome entry sequence (IRES), a polyadenylation sequence and a splice sequence.
In another aspect of the invention, the shuttle vector further comprises an origin of replication functional in a bacterial cell and preferably, a selectable gene functional in a bacterial cell. The shuttle vector may also comprise an origin of replication functional in a mammalian cell, and optionally, a selectable gene functional in a mammalian cell.
The present invention also provides methods for using the shuttle vectors provided herein. In one embodiment, heterologous nucleic acids flanked by regions identical to flanking regions of the insertion site within a shuttle vector are co-introduced to yeast with the shuttle vector and allowed to homologously recombine such that the heterologous nucleic acids are inserted into the shuttle vector by the yeast organism. In preferred embodiments, the heterologous nucleic acids are introduced to the yeast in a linear nucleic acid. The shuttle vector is then recovered and transferred to a mammalian cell for expression.
REFERENCES:
patent: 4783412 (1988-11-01), Bell
patent: 5741486 (1998-04-01), Pathak et al.
patent: 5962320 (1999-10-01), Robinson
patent: 6391582 (2002-05-01), Luo et al.
Camonis et al. “Of mice and yeast: versatile vectors which permit gene expression in both budding yeast and higher eukaryotic cells”Gene86(2):263-268 (1990).
Storck et al. “Rapid construction in yeast of complex targeting vectors for gene manipulation in the mouse” Nucleic Acids Research 24(22):4594-4596 (1996).
Lorens James
Luo Ying
Yu Pei Wen
Ketter James
Lahive & Cockfield LLP
Rigel Pharmaceuticals Inc.
Smith, Esq. DeAnn F.
Williams, Esq. Megan E.
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