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Cold Storage
Cell lines are routinely frozen to protect them from degradation
during preservation, but upon thawing, the membrane integrityof the cells is often disrupted. Studying the onset of these preservation injuries can be challenging due to the limited availability ofappropriate culture chambers for maintaining live cells at temperatures below 10°C during microscopic imaging. Writing in thisissue of BioTechniques, Sigot et al. at the Universidad Nacionalde Rosario (Rosario, Argentina) describe the construction andperformance of an inexpensive modular perfused chamber (MPC)
for imaging live cells at low temperatures using an inverted microscope. The MPC is composed of four lathed pieces of stainless steel
assembled to form an open cylinder. The chamber is autoclavableso cells may be grown directly inside it, or the chamber can holdcoverslips carrying cells cultured in other locations. Temperature
within the MPC is controlled using a standard thermal water
bath by circulating the water. When imaging at cold temperatures, condensation may form on the coverslip and interferewith the quality of image acquisitions, so the authors directeda stream of cool air onto the coverslip. The ability of the device

to aid in biological studies was demonstrated by following theuptake of quantum dots coupled to epidermal growth factor(EGF) in rat hepatocytes as they recovered from hypothermicpreservation. Activation of the EGF receptor results in clathrinmediated endocytosis via coated pits, a process inhibited by low
temperatures. Images were acquired from the endpoint of preservation, throughout rewarming of the cells, and for 40 min after
the cells reached physiological temperature. Internalization ofthe quantum dots was observed after 30 min at 37°C, indicating
restored endocytosis, basal recovery of the ATP pool, and viabilityof the recovering cells. This experiment shows the value of theMPC in evaluating the effect of cold storage on the recovery of
endocytic and energy-dependent transport in hepatocytes andshould also be useful in other studies of the onset of cold-induced
injury or to examine cellular processes that occur during coolingto preservation temperatures.
See “A modular perfused chamber for low- and normaltemperature microscopy of living cells” on page 251.

Lots More loxP
Site-specific recombination is an important
tool for various methods of genome
engineering. Two of the most commonly usedsite-specific recombination systems are Cre
recombinase, with its recognition site loxP,and Flp recombinase, with its recognition site
Frt. The combination of these two systems
has proven to be especially useful (e.g., forgenerating mouse conditional knockouts).
Increasing the relatively limited number ofsite-specific recombination systems would
facilitate the execution of more complicated genome engineering schemes involving
greater numbers of cassettes and targetsites. In this issue, S. Minorikawa and M.
Nakayama at the Kazusa DNA ResearchInstitute (Chiba, Japan) describe the application to genome engineering of their two
recently developed site-specific recombination systems, VCre/VloxP and SCre/SloxP.
These systems were isolated respectively fromVibrio and Shewanella and are members of
the same tyrosine recombinase family as
Cre and Flp. The VCre and SCre recombinases are both weakly homologous toCre, and their recognition sites, VloxP andSloxP, are different from each other and loxP.
These two novel systems had been previously demonstrated to not interact withthe Cre/loxP nor Flp/FRT systems. Here,the authors first showed that recombinasemediated cassette exchange (RMCE) could
be carried out with VCre, VloxP, and themutant recognition site Vlox2272, which,Vol. 50 | No. 4 | 2011

Predicted three-dimensional structures of the VCre and SCre recombinases compared to that of Cre recombinase.

like the lox2272 site, can only recombinewith itself and not with VloxP. Using adonor cassette flanked by a VloxP and aVlox2272 site, RMCE with an appropriate
recipient plasmid was easily induced in thepresence of VCre expression. The analogous
RMCE experiment using SCre, SloxP, andSlox2272 could also be easily done. Next,
they demonstrated that VCre/VloxP could
be combined with λ Red recombineering toengineer a BAC. A PCR cassette bearing ablasticidin resistance gene flanked by twoVloxP sites, as well as a puromycin gene, wasintroduced by recombineering into a human
Nanog BAC clone. Subsequent expression ofVCre caused the elimination of the blasticidin resistance gene flanked by VloxP sitesfrom the recombineered BAC in Escherichia

205

coli. These two new site-specific recombination systems should aid in expanding thescope and complexity of genome engineering
schemes.
See “Recombinase-mediated cassette
exchange (RMCE) and BAC engineering
via VCre/VloxP and SCre/SloxPsystems”
on page 235.
Selected and written by Patrick C.H. Lo,Ph.D. and Kristie Nybo, Ph.D.
BioTechniques 50:205 (April 2010)
doi 10.2144/000113643
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