Site-specific recombinase technology: Difference between revisions

Revision as of 10:51, 2 November 2014

Revision as of 09:50, 22 November 2014

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Common genetic engineering strategies require a permanent modification of the target genome. To this end great sophistication has to be invested in the design of routes applied for the delivery of transgenes. Although for biotechnological purposes random integration is still common, it may result in unpredictable gene expression due to variable transgene copy numbers, lack of control about integration sites and associated mutations. The molecular requirements in the stem cell field are much more stringent. Here, [[homologous recombination]] (HR) can, in principle, provide specificity to the integration process, but for eukarytoes it is compromised by an extremely low efficiency. Although meganucleases, zinc-finger- and transcription activator-like effector nucleases (ZFNs and TALENs) are actual tools supporting HR, it was the availability of site-specific recombinases (SSRs) which triggered the rational construction of cell lines with predictable properties. Nowadays both technologies, HR and SSR can be combined in highly efficient "tag-and-exchange technologies".<ref>{{cite journal |doi=10.1016/S1534-5807(03)00399-X |title=Talking about a RevolutionThe Impact of Site-Specific Recombinases on Genetic Analyses in Mice |year=2004 |last1=Branda |first1=Catherine S. |last2=Dymecki |first2=Susan M. |journal=Developmental Cell |volume=6 |pages=7–28 |pmid=14723844 |issue=1}}</ref>

Many [[site-specific recombination]] systems have been identified to perform these DNA rearrangements for a variety of purposes, but nearly all of these belong to either of two families, tyrosine recombinases (YR) and serine recombinases (SR), depending on their [[site-specific recombination|mechanism]]. These two families can mediate up to three types of DNA rearrangements (integration, excision/resolution, and inversion) along different reaction routes based on their origin and architecture.<ref name= "nern">{{cite journal |doi=10.1073/pnas.1111704108 |bibcode=2011PNAS..10814198N |title=Multiple new site-specific recombinases for use in manipulating animal genomes |year=2011 |last1=Nern |first1=A. |last2=Pfeiffer |first2=B. D. |last3=Svoboda |first3=K. |last4=Rubin |first4=G. M. |journal=Proceedings of the National Academy of Sciences |volume=108 |issue=34 |pages=1419814198–203 |pmid=21831835}}</ref>

The founding member of the YR family is the [[lambda integrase]], encoded by [[Bacteriophage| bacteriophage λ]], enabling the integration phage DNA into the bacterial genome. A common feature of this class is a conserved tyrosine nucleophile attacking the scissile DNA-phosphate to form a 3'-phosphotyrosine linkage. Early members of the SR family are closely related resolvase/invertases from the bacterial transposons Tn3 and γδ, which rely on a catalytic serine responsible for attacking the scissile phosphate to form a 5'-phosphoserine linkage. These undisputed facts, however, were compromised by a good deal of confusion at the time other members entered the scene, for instance the YR recombinases [[Cre recombinase|Cre]] and [[FLP-FRT recombination|Flp]] (capable of integration, excision/resolution as well as inversion), which were nevertheless welcomed as new members of the "integrase family". The converse examples are PhiC31 and related SRs, which were originally introduced as resolvase/invertases although, in the absence of auxiliary factors, integration is their only function. Nowadays the standard activity of each enzyme determines its classification reserving the general term "recombinase" for family members which, per se, comprise all three routes, INT, RES and INV:

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*http://www.knockoutmouse.org/

*{{cite journal | last1 = Emes | first1 = RD | last2 = Goodstadt | first2 = L | last3 = Winter | first3 = EE | last4 = Ponting | first4 = CP | year = 2003 | title = Comparison of the genomes of human and mouse lays the foundation of genome zoology | url = http://hmg.oxfordjournals.org/content/12/7/701.long | journal = Hum Mol Genet | volume = 12 | issue = 7| pages = 701–9 | doi=10.1093/hmg/ddg078 | pmid=12651866}}

[[Category:Genetic engineering]]