Target Discovery and Validation Reviews and Protocols

RNAi Libraries 135
pathways. Thus, several signaling proteins might work in concert to affect apoptosis
in HeLa S3 cells (30).
3.1. Construction of siRNA Expression Vectors
The siRNA library was constructed in pcPUR hU6 vector (iGENE
Therapeutics, Inc.). The vector contains a human U6 promoter, a puromycinresistance
gene, and BspMI cloning sites (Fig. 2).
3.1.1. Preparation of Vector DNA
1. Digest 3–5 µg of pcPUR hU6 plasmid with restriction enzyme BspMI.
2. Purify the cleaved vector from an agarose gel (0.8%) by using QIAquick gel
extraction kit.
3. Elute DNA in 30 µL of TE buffer and use 1 µL for ligation reaction.
3.1.2. Preparation of siRNA Oligonucleotides
1. Design and synthesize the required DNA oligonucleotides for targeting the
human genes that are selected to be included in the library (see Note 1). Once the
target sites are selected, for each site design two complementary oligonucleotides:
5′-cac c [sense sequence] gt gtg ctg tcc [antisense sequence] tt ttt-3′
and 5′-gca taa aaa [sense sequence] gg aca gca cac [antisense sequence]-3′
(see Notes 2 and 3).
2. Each pair of oligonucleotides should be annealed using the following conditions:
combine 5 µL of each oligonucleotide (100 µM), heat to 95°C for 2 min with thermal
cycler, cool to 72°C and then cool to 4°C for 2 h.
3. Dilute the annealed oligonucleotide to 200-fold with TE buffer.
3.1.3. Ligation and Transformation
1. Mix 1 µL of the annealed oligonucleotides, 1 µL of the purified DNA vector, and
1 µL of ligation high (the DNA ligase is included).
2. Incubate the mixture for at least 30 min at 16°C.
3. Transform the ligated DNA into E. coli host cells (DH5α) by using the following
protocol: mix the ligated plasmid and competent E. coli host cells (DH5α),
chill the mixture on ice for 5 min, heat shock the cells in hand for 30 s, and
then incubate on ice for 2 min. Plate the cells directly on preheated LB plates
with 100 µg/mL ampicillin. More than 90% of colonies should be positive
clones.
3.2. Cell Culture and Transient Transfection
1. One day before the transfection, plate HeLa S3 cells at 1 × 104 cells/100 µL in
DMEM supplemented with 10% fetal bovine serum and 1% antibiotic-antimyoctic
(see Note 4).
2. Wash the wells twice with PBS buffer.