Cloning 3.0?

Abstract

If cloning technologies were software releases, you could say that two major versions have been created to date. Version 1.0 would probably include the basic techniques of cloning: using plasmids, restriction enzymes, and so on. Intermediate releases, say 1.1 to 1.5 came about with the improvement of vectors to detect inserts and the conversion of lambda clones. Version 2.0 came into being when the polymerase chain reaction (PCR) was developed in the late 1980s. PCR technology provided methods to better refine the DNA molecules to be inserted. It allowed novel molecules to be created in vitro that would be difficult or impossible to produce any other way. What, then, can we expect to find in the next “release” of cloning technology? A group led by Steve Elledge at Baylor College of Medicine ([1][1]) may have just answered this question and, in the process, ushered in a novel set of molecular technologies for the post-genome era. One of the major tasks for a molecular biologist is moving known pieces of DNA from one cloning vector to another to create various fusions and clones. In the process, a researcher may use PCR to design joining segments, internal mutations, and other elements. All of these version 2.0 tasks rely on careful, custom construction by the genetic engineer. Many common DNA constructs (reporter fusions, GST fusions, myc-tag fusions) could be made more easily if a standard set of plasmids and manipulations were available. However, moving passenger DNA between template plasmids by restriction digests requires detailed knowledge of the passenger DNA, which is not always available. Also, as whole-genome research (for example, the Human Genome Project) progresses, cloning techniques to manipulate entire genomes become necessary. The method proposed by Q. Liu et al. ([1][1]), the Univector Plasmid-fusion System (UPS), generates a new approach to cloning. Basically, they devised a highly scalable system in which site-specific recombination is used to rapidly generate new fusion proteins. Instead of using restriction enzymes and DNA ligase to cut and paste DNA molecules, they used Cre recombinase, a protein that can catalyze the joining of two sequences that each have a defined 34-base pair (bp) sequence called loxP. They first intoduced their gene into the Univector (pUNI) in the form loxP-GENE-Kanr-Ori. The gene is placed in-frame with the open reading frame of loxP. Next, they made a collection of pHOST vectors that had a DNA sequence in the form Promoter-Tag-loxP-Ampr-Ori. They then mixed a pUNI clone in vitro with a pHOST plasmid and Cre protein. The result was a highly efficient, 20-min recombination reaction that produced a fusion plasmid joined at the loxP sites. The reading frame of the loxP site on pHOST, pUNI, and the Tag sequences are the same, so that fusion of pUNI and pHOST results in the creation of both a promoter-gene fusion and a protein-protein fusion. Furthermore, through genetic trickery, they devised a selection process through which only fusion plasmids grow upon transformation. The fusion reaction is so efficient that they can transfer a whole library in pUNI into a different vector without loss of representation. Thus, with a simple, systematic, and easily reproduced in vitro reaction, they were able to generate a large collection of gene fusions with little knowledge of the chosen gene's sequence. Once a gene is inserted properly into pUNI, there is no more need to worry about its sequence, its reading frame, or the recipient vector. There is also no need for more fragment isolations or ligations; only a 20-min reaction is required. This technique now allows the systematic manipulation of large gene sets or even complete sets of full-length complementary DNAs from a particular species (so-called “Unigene” sets). This capability will become even more important as we progress into the postgenome era of proteomics. The paper also contains a must-read collection of additional molecular biology tips and tricks that no graduate student should be without. 1. [↵][2]1. Q. Liu, 2. Z. M. Li, 3. D. Leibham, 4. D. Cortez, 5. S. J. Elledge , Curr. Biol. 8, 1300 (1998). 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