Target Discovery and Validation Reviews and Protocols

148 Sano and Taira
ribozymes may cleave messenger RNAs, and as a result, may change the cell
phenotype (Fig. 2). Thus, the identification of the target of the ribozyme allows
to the identification of the genes responsible for the specific phenotype.
Probably, one of the most important steps in this system is to isolate cells with
a phenotypic change caused by introducing the library. So far, several studies have
identified genes of interest that are responsible for many specific phenotypes
(6–16). A major advantage of the use of the ribozyme library is that no prior
sequence information is required and, thus, it allows the identification of previously
unidentified genes when large numbers of ribozymes are introduced into cells.
For successful experiment, we must carefully discriminate between cells with
phenotypic change and unchanged cells, because the recovery of plasmids from
unchanged cells increases the number of false positives. Thus, we must fully
consider the strategy for reducing such false positives prior to the introduction
of the library into cells.
For example, we identified genes responsible for cell migration by a library
of randomized hammerhead ribozymes (6). A line of highly invasive cancer
cells, HT1080 fibrosarcoma cells, was transfected with a plasmid vector harboring
a randomized hammerhead ribozyme library and subjected to a chemotaxis
assay in 12-µm-pore Transwell inserts with fibronectin as a chemo-attractant.
Although HT1080 cells normally migrate toward a chemo-attractant, some cells
did not migrate toward the chemo-attractant after treatment with the randomized
ribozyme library. Ribozymes were recovered from cells that remained in
the top chamber and the randomized regions of the ribozymes were sequenced
in order to search for the relevant sequences in databases (Fig. 3).
At the first step, thus, we must design a sophisticated strategy and optimize
the condition for separating positive cells. Here, we describe typical transfection
protocol for introducing a ribozyme library into cells. After transfection, cells
should be separated by induction of the selection pressure that one chooses.
1. The day before transfection, plate cells at 70–90% confluence in a 10-cm dish on
the day of transfection.
2. Mix 15 µL of LipofectAMINE 2000 with 750 µL of OptiMEM I and incubate
at room temperature for 5 min. Appropriate transfection regents can be used,
depending on the type of cells used.
3. Mix 15 µg of the plasmid DNA and 750 µL of OptiMEM I.
4. Combine the diluted DNA solution with the diluted LipofectAMINE 2000
reagent and incubate for 15 min. While complex formation takes place, wash cells
with phosphate-buffered saline (PBS) and replace with 8.5 mL of fresh cellgrowth
medium.
5. Apply the complexes to cells and incubate at 37°C for appropriate time (typically
24–48 h) in a CO 2 incubator.
6. Induce the specific selection-pressure to isolate the cells with the phenotypic change.