Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
2000-06-28
2003-04-22
Wang, Andrew (Department: 1639)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
C435S455000, C435S471000, C435S007310, C435S007320, C435S007210
Reexamination Certificate
active
06551828
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to recombinant DNA technology, nucleic acids, vectors and methods for use in a recombinational cloning or subcloning, and more specifically for constructing expression vectors by using recombination proteins in vitro or in vivo through site-specific recombination.
DESCRIPTION OF RELATED ART
Recombinant DNA technology, also called gene cloning or molecular cloning, is widely used to transfer genetic information, i.e. DNA, from one organism to another. A typical recombinant DNA experiment often follows the following procedure. First, the DNA (e.g., the cloned DNA, insert DNA, target DNA, or foreign DNA) from a donor organism is extracted, enzymatically cleaved (or cut/digested), and joined (ligated) to another DNA entity (e.g. a cloning vector) to form a new, recombinant DNA molecule (or cloning vector-insert DNA construct). Second, this cloning vector-insert DNA construct is transferred into and maintained within a host cell, such as transformation of a bacterial host cell by the construct. Third, those host cells that take up the DNA construct (transformed cells) are identified and selected from those that do not. In addition, if required, a DNA construct can be prepared to ensure that the protein product that is encoded by the cloned DNA sequence is produced by the host cell.
Accordingly, this traditional cloning methods using restriction enzymes and ligase can be time consuming, especially when a specific expression vector is required for transferring the target gene into a heterologous host cell, such as a mammalian cell. The specific expression vector may not contain matching restriction sites for the donor DNA. Extensive reengineering of the expression vector may be required to introduce the matching restriction sites into the vector so that the vector and the insert DNA can be ligated to produce the final construct. Alternatively, multiple restriction enzymes may have to be employed to generate an insert DNA having suitable restriction sites for ligation with the vector. In this case, reaction conditions for each restriction enzyme may differ such that it is often necessary to perform a few separate restriction digestion reactions to obtain the desired insert. Further, the efficiency of direct ligation between the vector and insert may be very low, especially between large fragments. As a result, the whole procedure is tedious, and the final yield of the correctly ligated construct can be low.
Site-specific recombination represents another useful method of recombinant DNA technology. This method employs a site-specific recombinase, an enzyme which catalyzes the exchange of DNA segments at specific recombination sites. Site-specific recombinases present in some viruses and bacteria, and have been characterized to have both endonuclease and ligase properties. These recombinases, along with associated proteins in some cases, recognize specific sequences of bases in DNA and exchange the DNA segments flanking those segments. Landy, A. (1993) Current Opinion in Biotechnology 3:699-707.
A typical site-specific recombinase is Cre recombinase. Cre is a 38-kDa product of the cre (cyclization recombination) gene of bacteriophage P1 and is a site-specific DNA recombinase of the Int family. Sternberg, N. et al. (1986) J. Mol. Biol. 187: 197-212. Cre recognizes a 34-bp site on the P1 genome called loxP (locus of X-over of P1) and efficiently catalyzes reciprocal conservative DNA recombination between pairs of loxP sites. The loxP site consists of two 13-bp inverted repeats flanking an 8-bp nonpalindromic core region. Cre-mediated recombination between two directly repeated loxP sites results in excision of DNA between them as a covalently closed circle. Cre-mediated recombination between pairs of loxP sites in inverted orientation will result in inversion of the intervening DNA rather than excision. Breaking and joining of DNA is confined to discrete positions within the core region and proceeds on strand at a time by way of transient phophotyrosine DNA-protein linkage with the enzyme. Other examples of site-specific recombination systems include the integrase/att system form bacteriophage &lgr; and the FLP/FRT system from the
Saccharomyces cerevisiae
2pi circle plasmid.
These site-specific recombination systems have been used in vivo to facilitate recombination between different vectors. Waterhouse et al. used an in vivo method to join light and heavy chains of an antibody. The light and heavy chains were cloned in different phage vectors between loxP and loxP 511 sites that were used to transform new
E. coli
cells. Waterhouse, P. et al. (1993) Nucleic Acid Res. 21:2265-2266. Cre acted on two parental molecules, one plasmid and another phage, in the host cells to produce four products in equilibrium: two different cointegrates (produced by recombination at either loxP or loxP511 sites), and two daughter molecules, one of which was the desired product. Schlake and Bode used an in vivo method to exchange expression cassettes at defined chromosomal locations, each flanked by a wild type and spacer-mutated FRT recombination site. Schlake and Bode (1994) Biochemistry 33:12746-12751. A double-reciprocal crossover was mediated in cultured mammalian cells by using the FLP/FRT system for site-specific recombination. Aoki et al. used a shuttle plasmid (pAdMCS) that carried a gene of interest, a loxP site, the adenoviral 5-LTR and packaging signal 0 to 1 mu, and a multiple cloning site. Aoki et al. (1999) Mol. Med. 5:224-231. The shuttle plasmid was linearized by a restriction enzyme Nhel and recombined with Clal-digested adenoviral cosmid in vitro. Cre recombinase produced the full-length recombinant adenoviral vector in vitro by an exchange of region distal to the loxP site linearized in these two molecules.
SUMMARY OF THE INVENTION
The present invention relates to compositions, kits, and methods for use in a recombinational cloning or subcloning. In particular, the present invention provides novel methods for constructing expression vectors by using site-specific recombinases in vitro. These method may be used for high throughput screening of genes, functional genomics and other human genome projects.
In one aspect, the present invention provides a double-stranded circular donor DNA for transferring a donor DNA sequence into expression vectors. The circular donor DNA comprises: a donor DNA sequence; a donor recombination site; at least one selectable marker, the circular donor DNA not including an origin of replication.
The donor DNA sequence may be any gene of interest or any synthetic DNA sequence which is needed to be transferred into an expression vector. For example the donor DNA segment may be a sequence derived from cDNA of a particular gene or one of the members of a cDNA library. The donor DNA may also be a genomic DNA that contains the coding region interrupted with non-coding sequences.
In another aspect, the present invention also provides a library of double-stranded circular donor DNAs that may be used for high throughput screening. The library of double-stranded circular DNA comprises: a donor DNA sequence which varies within a library of donor DNA sequences; a donor recombination site; and at least one selectable marker, the circular donor DNA not including an origin of replication.
The library of donor DNA sequences may be a library of cDNA or genomic DNA derived from any desirable sources. For example, the library of donor DNA sequences may be a cDNA library from single human chromosomes.
The circular donor DNA may further comprise a promoter sequence that controls expression of the donor DNA sequence. The promoter may be any array of DNA sequences that interact specifically with cellular transcription factors to regulate transcription of the downstream gene. The promoter may be derived from any organism, such as bacteria, yeast, insect and mammalian cells and viruses. Examples of the promoter include, but are not limited to,
E. coli
lac and trp operons, the tac promoter, the bacteriophage &lgr;&bgr;
L
promoter, bacterio
Chen Shirley
Friend Tomas
Protemation, Inc.
Wang Andrew
Wilson Sonsini Goodrich & Rosati
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