Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-11-28
2002-04-23
Horlick, Kenneth R. (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S477000
Reexamination Certificate
active
06376192
ABSTRACT:
BACKGROUND
1. Field of the Invention
The present invention is related to the field of molecular biology and more particularly to the field of screening DNA libraries such as genomic and cDNA libraries to isolate a desired gene, and to the field of construction of targeting vectors for use in targeting a chromosomal gene, wherein the targeting vectors are constructed utilizing homologous recombination in
E. coli.
2. Description of Related Art
The development of technologies for targeted gene disruption in mouse embryonic stem cells (ES cells) has profoundly shaped biological research and the technique is now routinely used in laboratories. As the human genome project comes to completion and the mouse genome project comes to center stage, the demand for knockout mice is certain to increase dramatically in hopes of defining functions of the large volume of genes discovered by whole genome sequencing. The production of knockout mice, however, is still a time consuming process. A number of molecular manipulations must be performed to build a knockout construct to target the gene of interest in ES cells. First, genomic clones of the gene must be isolated and characterized. Secondly, a knockout construct is built in which a positive selection marker (usually the neomycin or puromycin resistant gene) is flanked by genomic sequences of several kilobases and a negative selection marker (usually the thymidine kinase of herpes virus) is placed at one end followed by plasmid backbone. These manipulations can be a rate-limiting step for generation of a knockout mouse and can often inhibit the decision to make a genetically altered mouse.
To identify a genomic region of interest, prior methods used plaque hybridization using a radioactive probe, usually a cDNA from the gene of interest, to identify the lambda phage containing the homologous genomic region of interest. This is labor intensive requiring multiple rounds of purification to identify just a few homologous clones. In addition, since it is based on hybridization, it often can pick up related sequences such as psuedo-genes that have related but not strictly identical DNA sequences. Similarly, the methods to identify full length cDNAs from libraries also require plaque screens using hybridization and radioactive probes and multiple rounds of screening. Thus, the methods previously used to identify phage containing homologous genomic DNA and cDNA are both laborious and time consuming.
It was contemplated by the present inventors that the described processes may be simplified by taking advantage of homologous recombination in
E. coli.
Several recombination pathways have been identified in
E. coli
with the RecBCD pathway playing a major role in the double-strand break repair pathway. RecBCD encodes a helicase and a nuclease that unwind and degrade DNA to generate 3′single-stranded tails utilized by the RecA protein for invasion to initiate the recombination process. However, homologous recombination efficiency between a linear piece of DNA and the host chromosome is very low in
E.coli
cells that express wildtype RecBCD because the introduced linear DNA molecules are degraded rather efficiently before recombination has had a chance to proceed. It has been found that a short sequence, 5′-GCTGGTGG-3′, called a Chi site, can stimulate homologous recombination, as this short sequence is inhibitory to the nuclease function of RecBCD. In order for a linear DNA molecule to recombine with the host chromosome or a resident plasmid, either RecBCD must be inactivated or Chi sites must be present on the linear DNA. In a strain with a mutant RecBCD (JC8679[ ], for example), linear DNA can recombine with a host chromosome or plasmid with modest efficiency. Initial attempts by the inventors to utilize the mutant RecBCD strain were not ideal, however, because the recombination efficiency was too low to screen for single copy genes within the complexity of the mouse genome. There is still a need, therefore, for simple methods of screening a genomic library and constructing targeting vectors for use in the knock out of genes of interest in mammalian species, such as mice.
SUMMARY
The present disclosure overcomes drawbacks in the prior art by providing compositions and methods that simplify the screening of DNA libraries to select genes of interest through the use of homologous recombination in
E. coli
. A particular advantage of the present invention is that one can identify and select a gene of interest based on only about 60-100 bases of homology and can at the same time modify that gene fragment for use as a knockout targeting vector, for example. The invention is particularly useful in the screening of large libraries such as mammalian genomic libraries for the isolation of genomic copies of mammalian genes, for example, and in the construction of knockout targeting vectors. The invention is also useful for the screening of any DNA target, including cDNA libraries, BAC libraries, or cosmid libraries for various applications such as to extend partial sequences or to fill in sequence gaps in such libraries or genes within those libraries.
The advantages of the present compositions and methods arise from the ability to select a gene of interest from a DNA library based on the homology required for homologous recombination and simultaneously insert a positive selection marker into that gene so that only the targeted clones survive in the selection media. Using the compositions and methods described herein, one is thus able to isolate nucleic acid segments or clones of interest from a nucleic acid library via homologous recombination using regions of homology as small as about 60-100 base pairs (bp). By this description of the homology being about 60-100 base pairs, it is understood that this represents a minimum amount of sequence homology of about 56, 58 or 60 base pairs, but that much larger regions may be used, such as 200-500 or even several thousand bases or more of homology as desired by the practitioner. It is also understood that the regions of targeting homology are separated by the selection marker so that, in certain embodiments approximately one half the region of homology will appear at each end of the selectable marker region in the targeting construct. In certain preferred embodiments, then one may use regions of homology of about 26, 28, 30, 40, 50, 60, 75 or even 100 bases for each targeting region, making a total of about 50 or 60 to 200 bases of total homology.
Once the clones are selected and isolated, they can then be sequenced and used to construct complete genes or cDNA sequences, to fill gaps in sequence data, or even for genomic walking to obtain further sequence data. Furthermore, the compositions and methods of the present disclosure allow the screening of a library and production of a finished genetic targeting construct in less than a week, in contrast to months of work that are often required to produce such constructs using conventional methods.
Another aspect of the present disclosure is the use of recombination functions from the bacteriophage &lgr; in
E. coli
. The recombination function of &lgr; phage is carried out by two gene products, exo, a nuclease that acts progressively on double strand DNA to generate a 3′ single stranded overhang and beta, a single-strand DNA (ssDNA) binding protein capable of annealing complementary ssDNA strands. In certain preferred embodiments, the homologous recombination includes the inactivation of the
E. coli
RecBCD by a &lgr; phage gam gene product.
In the screening assays, one may insert a linear DNA fragment composed of a selectable marker flanked by regions of homology to the gene of interest into a library containing cell culture by electroporation and subsequent selection for the drug resistance marker. In the second type of assay one may also provide the recombinogenic fragment in vivo by placing the fragment encoding the selectable marker flanked by regions of homology into a specialized plasmid designed so that the fragment can be
Elledge Stephen J.
Li Mamie
Zhang Pumin
Baylor College of Medicine
Horlick Kenneth R.
Strzelecka Teresa
Vinson & Elkins
LandOfFree
Method for screening of DNA libraries and generation of... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Method for screening of DNA libraries and generation of..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Method for screening of DNA libraries and generation of... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2857887