构建shRNA表达质粒

onstruction of shRNA Expression Plasmids C

for Silkworm Cell Lines Using Single-Stranded DNA and Bst DNA Polymerase

HiromitsuTanaka

Abstract

ransfection of short hairpin RNA (shRNA) expression plasmids is conventionally performed for gene-speci Tfi c knockdown in cultured mammalian and insect cells. Here, I describe a simple method to synthesize an inverted repeat DNA in a U6 small nuclear RNA promoter-based parent vector using a single-stranded inverted repeat DNA and Bst DNA polymerase. The shRNA expression plasmids constructed by this method were con fi rmed to promote ef fic ient RNA interference knockdown in silkworm cell lines. This method may be useful for constructing a relatively large number of shRNA expression plasmids.

Key words: shRNA expression plasmids , Bst DNA polymerase , Single-stranded inverted repeat DNA ,

Silkworm cell line

1.Introduction

osttranscriptional silencing by RNA interference (RNAi) is widely P

used as a technique for suppressing the expression of speci fic genes

4 ). A conventional procedure for inducing in many organisms (1 –

RNAi knockdown in cultured mammalian and insect cells is the direct transfection of 21–23 nucleotides (nt) of small interfering

5, 6 ) or over-expression of short hairpin RNA RNA (siRNA) (

(shRNA) composed of 19–29 nt of stem regions and 4–23 nt of loop sequences by the transfection of an RNA polymerase III-8 ) . dependent promoter-driven shRNA expression plasmid (7 ,

Knockdown by transfection of an shRNA expression plasmid has

) . First, some advantages over knockdown by siRNA transfection (9

the RNAi effect may be more stable because of the sustained production of shRNA. Second, the transfected cells can be selected by antibiotics when the shRNA expression plasmid possesses

Debra J. Taxman (ed.), siRNA Design: Methods and Protocols, Methods in Molecular Biology,vol. 942,DOI 10.1007/978-1-62703-119-6_18, © Springer Science+Business Media, LLC 2013

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348H. Tanaka

antibiotic-resistance genes. Furthermore, inducible shRNA expression is available. In general, shRNA expression plasmids can be generated by two methods. One method is the insertion of a double-stranded inverted repeat (IR) DNA that is obtained by annealing of two complementary oligonucleotides into a parent

) . The second method is a polymerase chain reaction (PCR)-vector (9

based strategy in which the promoter sequence serves as the template ) . We developed another simple method to create IR DNA in the (1 0

parent vector using a single-stranded DNA possessing a short hairpin structure and Bst DNA polymerase, which has strand displacement

11 ) . This method comprises the following steps: (a) linear-activity (

ization of the plasmid with the 5 ¢ end of one terminus dephosphory-lated by treating stepwise with one restriction enzyme, alkaline phosphatase, and then a second restriction enzyme; (b) ligation of a hairpin oligonucleotide to one end of the linear plasmid; (c) execu-tion of the strand displacement reaction by Bst DNA polymerase; and

1 ). This method reduces (d) self-ligation of the linear plasmid (Fig.

the cost of unique oligonucleotides compared with the conventional method. Therefore, it is useful for constructing relatively large num-bers of shRNA expression plasmids. We further demonstrated that the shRNA expression plasmid constructed by this method effec-) . tively induces target-speci fi c RNAi a silkworm cell line (1 1

2.Materials

2.1.Oligonucleotide

Annealing

1. Synthesized oligonucleotides: 53 mer; 21 mer of IR structure

separated by 11 mer of spacer DNA. These oligonucleotides may be obtained from most custom oligonucleotide-synthesizing facilities and companies. For a discussion of the oligonucleotide design, see Notes 1 –5 . Store at −20°C. , 2. 10× M buffer: 100 mM Tris–HCl (pH 7.9), 100 mM MgCl 2500 mM NaCl, and 10 mM DTT. Store at −20°C. 3. Ultrapure water: Milli Q grade; sterilized by autoclaving.

2.2.Construction

of shRNA Expression Plasmids

1. Parent plasmid for constructing an shRNA expression plasmid:

We used pIEx-4-BmU6M, which contains the enhancer and promoter region between Sma I and Nco I. Multicloning sites between the Nco I and Dra III sites of pIEx-4 were substituted by 467 bp of the promoter region of the Bombyx mori U6-2

12 ) and the sequence “5 ¢ - CCATGGsmall nuclear RNA gene (

CTGCAG AGGCCT TTTTCACTAAGTG-3 ¢ ” (underlining indicates the Nco I site; bold letters indicate the Stu I site), respectively. 2 . Restriction endonucleases: Nco I and Stu I at 10 U/ m L. Store at −20°C.

18 shRNA Construction for Silkworms Using Bst DNA Polymerase349

a

B. mori U6-2 promoterCCATGGCTGCAGAGGCCTTTTTCACTAAGTGGGTACCGACGTCTCCGGAAAAAGTGATTCACIE1 terminatorSmaINcoIStuIpIEx-4-BmU6MDraIllb

pIEx-4-BmU6MNcoI digestion

Alkaline phosphatase treatmentStuI digestionOH 3’OH 5’5’ P3’ OH

Short hairpin oligonucleotideOH 3’OH 5’OH 3’OH 5’

OH 5’OH 3’OH 5’3’ OHOH 5’OH 3’5’ OHOH 5’3’ OH

Nick3’ OHLigation

Bst DNA polymerase treatment

T4 polynucleotide kinase treatmentSelf-ligation

Fig. 1. The structure of pIEx-4-BmU6M and the procedure for construction of an shRNA expression plasmid. ( a ) Diagram of pIEx-4-BmU6M. The nucleotide sequences possessing the Stu I recognition site ( Stu I) and a T cluster were inserted into the Nco I and Dra III sites of pIEx-4 (Novagen); the enhancer and promoter region between the Sma I and Nco I sites of pIEx-4 was replaced by 467 bp of a promoter region of Bombyx mori U6-2 small nuclear RNA gene ( black box ). Gray box indicates the terminator region from the Autographa californica nucleopolyhedrovirus-derived immediate early 1 gene. ( b)Strategyto create the IR DNA in pIEx-4-BmU6M. A short hairpin oligonucleotide is ligated with the Stu I-digested blunt end of linear pIEx-4-BmU6M. Bst DNA polymerase extends the 3 ¢ end of the Nco I-digested terminus and 3 ¢ end at the nick followed by the displacement of the 5 ¢ portion of the hairpin oligonucleotide. Kinase reaction and self-ligation yield a circular shRNA expressionplasmid.

3. 10× H buffer: 500 mM Tris–HCl (pH 7.9), 100 mM MgCl , 2

1 M NaCl, and 10 mM DTT. Store at −20°C. 4. Alkaline phosphatase: 10 U/ m L. Store at −20°C.

5 . CIA: 24:1 (v/v) mixture of chloroform and isoamyl alcohol. 6 . Phenol/chloroform: 1:1 (v/v) mixture of Tris–HCl (pH 8.0)

buffered phenol and CIA. Store at 4°C. 7 . Ethanol: 100% and 70% (v/v) solution.

350H. Tanaka

2.3.Confirmation

of Insert Size by Colony PCR

8. 3 M Sodium acetate (pH 5.2): Sterilized by autoclaving. 9. T

E buffer: 10 mM Tris–HCl (pH 8.0) and 1 mM EDTA. Sterilized by autoclaving.

10. 1 0× M buffer: 100 mM Tris–HCl (pH 7.9), 100 mM MgCl 500 mM NaCl, and 10 mM DTT. 2, 11. D

NA Ligation Kit Mighty Mix: Available from Takara Bio. Store at −20°C. 12. 5 0× TAE: 2 M Tris–acetate, 50 mM EDTA.

13. A garose gels: Electrophoresis grade agarose in 1× TAE. 14. W

izard SV Gel and PCR Clean-Up System: Available from Promega. 15. B st DNA polymerase large fragment and 10× ThermoPol Reaction Buffer: Bst DNA polymerase at 8 U/ m L and 10× ThermoPol Reaction Buffer at 200 mM Tris–HCl (pH 8.8),

100 mM KCl, 100 mM (NH ) SO , 20 mM MgSO , and 1% Triton X-100. Available from New England Biolabs. Store at 4244

−20°C. 1 6. 1

0 mM dNTP mixture: A mixture in water that contains 10 mM of each deoxyribonucleoside triphosphate. Store at −20°C. 1 7. T

4 polynucleotide kinase and 5× kinase buffer: T4 polynucle-otide kinase at 10 U/ m L and 5× buffer at 50 mmol/L

Imidazole–HCl (pH 6.4), 18 mM MgCl , 5 mM DTT, 6% (w/v) PEG6000. Store at −20°C. 2

18. 2 mM ATP: Store at −20°C.

19. C ompetent Escherichia coli : We successfully used both the Sure2 Supercompetent Cells (Stratagene) and DH5 a (Takara Co. Ltd) strains. Store at −80°C. 20. 2

× YT agar plate: To make 1 L, add 16 g of polypeptone, 10 g of yeast extract, 5 g of NaCl, and 15 g of agar to 900 mL of water. Fill to 1 L with water and autoclave. After cooling, add ampicillin to a fi nal concentration of 100 m g/mL. Pour into plates and store the plates at 4°C.

1 . F

orward and reverse primers: Dilute each synthetic oligonucle-otide to 10 m M with water. Store at −20°C. 2 . 1

0× PCR buffer: 100 mM Tris–HCl (pH 8.3), 500 mM KCl, and 15 mM MgCl 2. Store at −20°C. 3. T aq polymerase (5 U/mL): Store at −20°C.

4 . 2

× YT medium: 1.6% polypeptone, 1.0% yeast extract, and 85 mM NaCl. Sterilize by autoclaving.

18 shRNA Construction for Silkworms Using Bst DNA Polymerase351

3.Methods

3.1.OligonucleotideAnnealing

3.2.Construction

of shRNA Expression Plasmids

1. O

ligonucleotides were suspended in water to a concentration of 100 pmol/ m L. 2. M ix 32 m L of oligonucleotide solution (100 pmol/ m L), 32 m L of 10× M buffer, and 40 m L of water in a 0.2 mL tube. 3 . H

eat at 95°C for 5 min and gradually cool to 30°C (1–2°C/min). Annealed oligonucleotides should form a hairpin structure. 4 . S

tore at −20°C if the annealed oligonucleotides are not to be used immediately.

T he construction method using pIEx-4-BmU6M (1 1 ) is as follows: 1. D igest 10 m g of pIEx-4-BmU6M with 25 units of Nco I at

37°C for 1–12 h in a 400 m L reaction volume containing 40 m L of 10×H buffer. Heat DNA at 65°C for 5 min to inactivate the enzymes. 2 . A dd 2 m L of alkaline phosphatase and incubate the solution at 37°C for 30 min.

3 . E

xtract the reaction solution with phenol/chloroform and then CIA. Add 1 mL of absolute ethanol and 40 m L of 3 M sodium acetate to the upper phase solution. Centrifuge for 12,000 × g at 4°C for 10 min. Discard the supernatant, wash the pellet in 70% ethanol, and recentrifuge for 5 min. Dissolve the pellet with 357 m L of water and then add 40 m L of 10× M buffer and 2 m L of Stu I. Incubate at 37°C for 1–12 h. Extract the reaction solution with phenol/chloroform and then CIA. Precipitate the reactant DNA with ethanol. The pellet is dis-solved with TE buffer at a concentration of 0.25 m g/ m L. The product can be stored at −20°C. 4 . M ix 1.5 m L of linear plasmid, 1 m L of annealed oligonucle-otide, 5 m L of DNA Ligation Kit Mighty Mix, and 2.5 m L of water. Incubate at 16°C for 30 min. 5 . L oad 10 m L of ligated DNA solution onto a 1% agarose gel in 1× TAE gel running buffer. After electrophoresis is performed, remove the desired bands from the gel. See Note 6 . 6 . R

ecover DNA from the gel slice using a Wizard SV Gel and PCR Clean-Up System according to the instruction manual. Finally, elute DNA with 50 m L of water. 7 . M ix 43 m L of recovered DNA, 5 m L of 10× ThermoPol Reaction Buffer, 1 m L of 10 mM dNTP mixture, and 1 m L of B

st DNA polymerase. Incubate at 50°C for 2 min, and then move to 62.5°C for 30 min. 8. E

xtract the reaction solution with phenol/chloroform and then CIA. Precipitate the reactant DNA with ethanol. Dissolve

352H. Tanaka

the pellet with 43 m L of water. Add 11.3 m L of 5× kinase buffer, 1 m L of 2 mM ATP, and 1 m L of T4 polymerase kinase. Incubate at 37°C for 30 min.

9. Extract the reaction solution with phenol/chloroform and

then CIA. Precipitate the reactant DNA with ethanol. Dissolve the pellet with 20 m L of water. 10. Mix 5 m L of reactant solution and 5 m L of DNA Ligation Kit Mighty Mix. Incubate at 16°C for 30 min. 11. Transform 10 m L of ligation reaction into 100 m L of a compe-tent strain of E. coli . Plate the appropriate amount of cells onto 2× YT agar plates. Incubate at 37°C overnight.

3.3.Confirmation

of Insert Size by Colony PCR

1 . Prepare the PCR reaction mixture. For 500 m L, mix 50 m L of

10× PCR buffer, 10 m L of 10 mM dNTP mixture, 10 m L of each of forward and reverse primers (10 m M), and 5 m L of Taq polymerase. Then, bring to 500 m L with water. Dispense 10 m L of the PCR reaction mixture to each 0.2 mL PCR tube. 2. Pick each colony with a sterile toothpick, and swirl it into the

PCR reaction mixture in a tube.

3. Place each PCR tube in a thermal cycler. Heat at 95°C for

2 min, and then subject to 35 cycles as follows:

Denaturation 95°C for 0.5 min Primer annealing Primer extension 5 0–55°C for 0.5 min 7 2°C for 0.5–3 min 4. Run on a 1.5–2% agarose gel to analyze the insert size (See

Notes 7 and 8 ). 5. Culture positive colonies in 2 mL of 2× YT medium at 37°C

overnight. 6. Prepare a plasmid from the cultured E. coli , and con fi rm that the nucleotide sequence of the inserted DNA is correct.

4.Notes

1. GC contents in the stem region should be less than 55%; the

extension by Bst polymerase may not be completed if the GC contents are higher. 2. Stretches of four or more T nt should not be included in the stem

region because RNA polymerase III may terminate transcription by recognizing it as a terminator in the transfected cells. 3. For the spacer sequence of oligonucleotides, we used “5-GTGT

GCTGTCC-3 ¢ ,” which was derived from human microRNA

18 shRNA Construction for Silkworms Using Bst DNA Polymerase353

mir26b and has been reported to be an effective spacer sequence

14 ) . We con fi rmed in mammalian and Drosophila cell lines (1 3,

that the shRNA possessing this spacer sequence also effectively knocked down the target gene in silkworm cells. Furthermore, we con fi rmed that a randomly designed spacer “5 ¢ -AGTCCAACAGG-3 ¢ ” functioned ef fi ciently in the silk-worm cell lines. 4. shRNA with 19 or 17 nt stem regions were inef fic ient in silk-11 ) . Therefore, the length of the stem region worm cell lines (

should be at least 21 nt. 5 . In general, RNAi ef fi ciency in the cells is known to depend on the sequence of the stem region, and only approximately 30% of random siRNAs have been reported to show highly effective

15 ) . However, in our RNAi in cultured mammalian cells (

experiment, six of eight shRNA expression plasmids—each having randomly designed nucleotide sequences at the stem region—suppressed the expression of the reporter gene by

) . Another two constructs more than 95% in silkworm cells (1 1

also showed 75–80% reductions. These results suggest that sequence preference in silkworm cell lines is much lower than that in mammalian cell lines. 6. Agarose gel electrophoresis should be performed after ligation

of a hairpin oligonucleotide to the linear plasmid to remove

2 ). free hairpin oligonucleotides (Fig. 7. IR DNA-inserted plasmids can be easily distinguished from

3 ). However, incomplete empty plasmids by colony PCR (Fig.

IR DNA is sometimes inserted into the vector. Therefore, con fir mation of the nucleotide sequence of each plasmid is necessary. 8 . An examination of nine constructions using oligonucleotides

with 21 nt of stem regions and 11 nt of the spacer sequence “5 ¢ -GTGTGCTGTCC-3 ¢ ” revealed that 20–70% of the trans-formed clones contained correctly sized inserts by colony PCR. The ef fi ciency of creating an expected DNA insert in the plas-mid would be dependent on the nt sequences of the stem region. Analysis of nt sequences revealed that 68% of the recombinant clones possessing correctly sized inserts had cor-rect nt sequences, and that one additional nt was created in 80% of the clones at the junction between the Nco I site and the

) . oligonucleotide (1 1

Acknowledgments

his work was supported by a grant from Promotion of Basic T

Research Activities for Innovative Biosciences (PRO-BRAIN).

354H. Tanaka

Fig. 2. Agarose gel electrophoresis to separate the short hairpin oligonucletotide-ligated plasmid and free oligonucleotides. The short hairpin oligonucleotide-ligated plasmid ( arrow)is recovered by Wizard SV Gel and the PCR Clean-Up System. M; 1 kb DNA ladder.

Fig. 3. Con fi rmation of insert size in pIEx-4-BmU6M by colony PCR. Colony PCR was per-formed in a total volume of 10 m L that contained 200 nM of each forward “5 ¢-TGTAAAGTCGAGTGTTGTTGTA-3¢ ” and reverse “5 ¢-CAAAACCCCACACCAACAAC-3¢”primer. In this experiment, an oligonucleotide, “5 ¢-TCATTCCTGAAGACAGCTGAGGTGTGCTGTCCCTCAGCTGTCTTCAGGAATGA-3¢ ,” was used for construction of an shRNA expres-sion plasmid. Two different sizes of bands were detected. The band that was ampli fi ed from empty plasmids (Lines 3 and 5) was 221 bp long, and the band that was ampli fi ed from shRNA plasmids was 275 bp long (Lines 1, 2, 4, 6, 7, 8, 9, 10, 11). Nucleotide sequences of these plasmids were con fi rmed. C; pIEx-4-BmU6M. M; 100 bp DNA ladder.

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