C hapter 18 C onstruction of shRNA Expression Plasmids
for Silkworm Cell Lines Using Single-Stranded
DNA and B st DNA Polymerase
Hiromitsu Tanaka
Abstract
T ransfection of short hairpin RNA (shRNA) expression plasmids is conventionally performed for gene-speci fic 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 B st DNA polymerase. The shRNA expression plasmids constructed by this method were con fir med 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.
K ey words:s hRNA expression plasmids ,B st DNA polymerase ,S ingle-stranded inverted repeat DNA , S ilkworm cell line
1.Introduction
P osttranscriptional silencing by RNA interference (RNAi) is widely
used as a technique for suppressing the expression of speci fic genes
in many organisms (1–4). A conventional procedure for inducing
RNAi knockdown in cultured mammalian and insect cells is the
direct transfection of 21–23 nucleotides (nt) of small interfering
RNA (siRNA) (5, 6)or over-expression of short hairpin RNA
(shRNA) composed of 19–29 nt of stem regions and 4–23 nt of
loop sequences by the transfection of an RNA polymerase III-
dependent promoter-driven shRNA expression plasmid (7, 8).
Knockdown by transfection of an shRNA expression plasmid has
some advantages over knockdown by siRNA transfection (9). First,
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
vector ( 9
) . The second method is a polymerase chain reaction (PCR)-based strategy in which the promoter sequence serves as the template
( 10
) . We developed another simple method to create IR DNA in the parent vector using a single-stranded DNA possessing a short hairpin
structure and B st DNA polymerase, which has strand displacement
activity (
11 ) . This method comprises the following steps: (a) linear-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 B st DNA polymerase; and
(d) self-ligation of the linear plasmid (Fig.
1 ). This method reduces 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 ( 11
) . 1. S ynthesized 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 N otes 1 – 5 . Store at −20°C.
2. 10× M buffer: 100 mM Tris–HCl (pH 7.9), 100 mM MgCl 2 , 500 mM NaCl, and 10 mM DTT. Store at −20°C.
3. U ltrapure water: Milli Q grade; sterilized by autoclaving.
1. P arent plasmid for constructing an shRNA expression plasmid: We used pIEx-4-BmU6M, which contains the enhancer and promoter region between S ma I and N co I . Multicloning sites
between the N co I and D ra I II sites of pIEx-4 were substituted
by 467 bp of the promoter region of the B ombyx mori U6-2
small nuclear RNA gene (
12 ) and the sequence “5 ¢ - C CATGG C TGCAG A GGCCT T TTTCACTAAGTG-3 ¢ ” (underlining
indicates the N co I site; bold letters indicate the S tu I site),
respectively.
2. R estriction endonucleases: N co I and S tu I at 10 U/ m L . Store
at −20°C.
2.Materials
2.1.Oligonucleotide
Annealing 2.2.Construction
of shRNA Expression
Plasmids
349
reaction buffer18 shRNA Construction for Silkworms Using Bst DNA Polymerase 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. A lkaline phosphatase: 10 U/ m L . Store at −20°C.
5. C IA: 24:1 (v/v) mixture of chloroform and isoamyl alcohol.
6.
P henol/chloroform: 1:1 (v/v) mixture of Tris–HCl (pH 8.0) buffered phenol and CIA. Store at 4°C.
7. E thanol: 100% and 70% (v/v) solution.
5’ P 3’ OH
Nco I digestion Alkaline phosphatase treatment
Stu
OH 3’
OH 5’
Ligation
OH 5’
Bst DNA polymerase treatment
5’ OH
3’ OH T4 polynucleotide kinase treatment
Self-ligation
a b F ig. 1. T he structure of pIEx-4-BmU6M and the procedure for construction of an shRNA expressi
on plasmid. ( a ) Diagram of pIEx-4-BmU6M. The nucleotide sequences possessing the S tu I recognition site ( S tu I ) and a T cluster were inserted into the N co I and D ra I II sites of pIEx-4 (Novagen); the enhancer and promoter region between the S ma I and N co I sites of pIEx-4 was replaced by 467 bp of a promoter region of B ombyx mori U6-2 small nuclear RNA gene ( b lack box ). Gray box indicates the terminator region from the A utographa californica nucleopolyhedrovirus-derived immediate early 1 gene. ( b )Strategy to create the IR DNA in pIEx-4-BmU6M. A short hairpin oligonucleotide is ligated with the S tu I -digested blunt end of linear pIEx-4-BmU6M. B st DNA polymerase extends the 3 ¢ end of the N co 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 expression plasmid.
350H. Tanaka
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. 10× M buffer: 100 mM Tris–HCl (pH 7.9), 100 mM MgCl 2 , 500 mM NaCl, and 10 mM DTT. 11. D NA Ligation Kit Mighty Mix: Available from Takara Bio. Store at −20°C. 12. 50× 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: B st 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 4 ) 2 S O 4 , 20 mM MgSO 4 , and 1% Triton X-100. Available from New England Biolabs. Store at −20°C. 16. 10 mM dNTP mixture: A mixture in water that contains 10 mM of each deoxyribonucleoside triphosphate. Store at −20°C. 17. 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 2 , 5 mM DTT, 6% (w/v) PEG6000. Store at −20°C. 18. 2 mM ATP: Store at −20°C. 19. C ompetent E scherichia 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 n al 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. 10× 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/m L): Store at −20°C.
4. 2× YT medium: 1.6% polypeptone, 1.0% yeast extract, and
85 mM NaCl. Sterilize by autoclaving.
2.3.Con firmation
of Insert Size
by Colony PCR
351
18 shRNA Construction for Silkworms Using Bst DNA Polymerase 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 ( 11 ) is as follows: 1. D igest 10 m g of pIEx-4-BmU6M with 25 units of N co 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 S tu 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 N ote 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 3.Methods
3.1.Oligonucleotide
Annealing
3.2.Construction
of shRNA Expression
Plasmids
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