Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
1999-02-02
2002-01-01
Guzo, David (Department: 1636)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S235100, C435S320100, C435S471000, C435S472000, C435S475000, C435S243000, C435S252330, C435S252300, C536S023100
Reexamination Certificate
active
06335185
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to methods for the generation of lambda (&lgr;) or P1 bacteriophage vectors useful in targeted mutagenesis of eukaryotic cells and the expression of genes and proteins, methods for the identification of a &lgr; or P1 bacteriophage vector having a desired nucleic acid from an assortment or library of bacteriophage each having a different nucleic acid insert and the use of such vectors in gene targeting and the expression of genes and protein.
2. Description of the Related Art
The present invention provides a method for the construction of a &lgr; or P1 bacteriophage vector using bacteriophage/plasmid recombination and selection for double-crossover bacteriophage recombinants.
Vectors have traditionally been generated through restriction enzyme digestion of the vector and religation with the desired target nucleic acid. In general, two types of problems are encountered in the construction of vectors by this method. First, plasmid vectors may be undesirable because specific eukaryotic genomic regions can undergo rearrangements in plasmid vectors. Therefore genomic regions may be difficult to clone either on their own or in combination with eukaryotic selectable marker genes, such as neo or tk. Secondly, larger sizes of target DNA sequences are desirable. However, the larger the DNA sequence, the more restriction enzyme sites present in the DNA. For cloning purposes, suitable restrictions sites are low frequency sites located on either side of the target nucleic acid sequence. Therefore, because of the large number of restriction enzyme sites in large genomic fragments, the use of such fragments in vectors means that there is often very few or no suitable restriction sites for inserting foreign DNA fragments, such as positive selectable marker genes or small mutations.
Accordingly, it would be desirable to develop a method for the generation of vectors capable of accepting large genomic fragments without rearrangement and without the need for suitable restriction enzyme sites.
Bacteriophage/plasmid recombination has been used to screen and isolate targeted &lgr; phage from genomic libraries (15, 17). For example, a &lgr; genomic library (bearing amber mutations) was passaged over a rec
+
bacterial strain bearing a small supF (amber suppressing) recombination plasmid having sequence homologous to the desired gene. Homology in the recombination plasmid, usually derived from a cDNA sequence, directed the plasmid to integrate into the phage by a single crossover, thereby generating supF bearing phage recombinants capable of growing on a suppressor free (sup
0
) host. Depending on homology length, the recombination plasmid can integrate at a frequency of ~10
−2
. One of the difficulties with this method of bacteriophage/plasmid recombination is that it generated single cross-over recombinants. Single cross-over recombinants are generally considered undesirable because of the presence of plasmid sequences and the partial duplication of the target nucleic acid.
Accordingly, it would be beneficial to develop a method for the identification of a recombinant bacteriophage from a library through plasmid/phage recombination which method resulted in the isolation of the original bacteriophage without the insertion of the plasmid nucleic acid sequences.
Eukaryotic gene targeting involves the selection for homologous recombination events between DNA sequences residing in the genome of a eukaryotic cell or organism and newly introduced DNA sequences. This provides a means for systematically altering the genome of a eukaryotic cell or organism. For mammalian systems, laboratories have reported the insertion of exogenous DNA sequences into specific sites within the mammalian genome by way of homologous recombination. For example, targeted mutagenesis allows specific mutations to be engineered into the mouse germline via homologous recombination of exogenously-altered DNA in embryonic stem (ES) cells (1, 2). Using this technology, the function of any cloned gene may be examined by its disruption in mice. Thus, gene targeting is a critical experiment in molecular medicine, and is used, for example, to mimic human mutations in the mouse for the generation of experimental therapeutic models (3).
The original and still the most prevalent gene targeting approach ,“the knockout”, uses a replacement plasmid vector to direct a positive selectable marker (i.e. neomycin resistance gene) into a specific chromosomal location via either double-reciprocal exchange or gene conversion (4). Positive-negative selection vectors have been used for gene targeting (26). Many sophisticated variations on this original technique have become available, including the generation of point mutations, deletions and translocations and gene substitutions (5-9). Further, the application of cre recombinase from bacteriophage P1 allows additional genomic alterations at loxP target sequences following gene targeting so that mutations can be made tissue- or development-specific (10).
Although targeted mutagenesis provides a powerful tool for the analysis of gene function, it is a complex and time-consuming procedure. While methods of improving the efficiency of generating targeted ES cell lines (11) and mutant mice (12) have become available, little has been done to streamline the construction of the targeting vector. Currently the rate determining step in any gene targeting experiment is the construction of the targeting vector.
Accordingly, there is a need to develop a method to generate targeting vectors which does not require as much cumbersome restriction enzyme methodology and yet would yield targeting vectors which are efficient in inserting larger fragments of the modified nucleic acid into the desired site in the eukaryotic cell.
Further advantages of the present invention will become apparent from the following description of the invention with reference to the attached drawings.
SUMMARY OF THE INVENTION
This invention describes how double-crossover bacteriophage generated by bacteriophage/plasmid recombination can be selected through the use of double-crossover selectable markers present on the plasmid vector. The present invention is directed to the generation of &lgr; or P1 bacteriophage vectors using this method. The present invention is also directed to a method for screening a &lgr; or P1 bacteriophage library for the identification of a recombinant bacteriophage having the desired target sequence and to the generation of bacteriophage targeting vectors.
One aspect of this invention is directed to a method for generating recombinant &lgr; or P1 bacteriophage vectors, which method comprises
(a) providing a &lgr; or P1 bacteriophage nucleic acid sequence comprising a first target nucleic acid sequence;
(b) providing a plasmid comprising a nucleic acid sequence encoding a second modified target nucleic acid sequence, and a double-crossover selectable marker gene wherein the second modified target nucleic acid sequence is substantially homologous over a portion of its length to the first target nucleic acid sequence;
(c) contacting the bacteriophage and the plasmid under conditions such that homologous recombination between the first target nucleic acid sequence and the second target nucleic acid sequence occurs;
(d) selecting for double-crossover recombinant bacteriophage by placing the bacteriophage from step (c) under conditions such that bacteriophage having the double-crossover selectable marker are unable to replicate and isolating the double-crossover recombinant bacteriophage. The double-crossover selectable marker gene may be gam where the &lgr; recombinant bacteriophage is grown in a P2 lysogenic bacterial cell. The double-crossover selectable marker may be any large nucleic acid sequence where the &lgr; or P1 recombinant bacteriophage is placed under a size restriction, such as a requirement to be packaged in a viral coat or particle.
In this method, the plasmid may further comprise a prokaryotic positive selectable ma
Rancourt Derrick E.
Tsuzuki Teruhisa
Burns Doane , Swecker, Mathis LLP
Guzo David
Leffers, Jr. Gerald G.
University Technologies International Inc.
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