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Date: 19 April 2014
New Cost-effective method for gene silencing  

Topic Name: New Cost-effective method for gene silencing
Category: Genetic Engineering
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Research persons: Dr. Frank Buchholz’s group

Location: Max Planck Institute of Molecular Cell Biology and Genetics,Pfotenhauerstr. 108,01307 Dresden,, Germany


New Cost-effective method for gene silencing

Nearly a decade ago, now-Nobel laureates Craig Mello and Andrew Fire
discovered that they could insert short RNA molecules into worms and shut down
specific genes. Today, scientists routinely use this powerful method, termed RNA
interference, to study the functions of specific genes in mammalian systems.
In order to conduct these experiments, scientists generally rely on chemical
synthesis of RNA molecules, which can be quite costly. A freely accessible
article from this month’s release of
Cold Spring Harbor Protocols
 addresses this problem; it describes
a cost-effective approach for generating silencing RNAs, called esiRNAs, to
efficiently target virtually any gene in mammalian cells.
 describes how to enzymatically generate RNA molecules in
vitro, using the cloned gene of interest as a template. The RNA molecules are
then randomly cleaved into short fragments, purified, and used in RNA
interference experiments.
The procedure was developed by Dr. Frank Buchholz’s group at the Max Planck
Institute of Molecular Cell Biology and Genetics (Germany), and can be used to
generate large sets of esiRNA libraries to be applied to large-scale studies of
gene function .
Also highlighted in Cold Spring Harbor Protocols this month is an
article that describes how to culture thymus cells from

fetal mice
. The thymus is the organ where T-cells—a principal component of
the immune system in vertebrates—proceed through a strictly coordinated
maturation process before being released into the bloodstream. Fetal thymus
organ culture is the only system available for studying the complete program of
T-cell maturation in vitro, and the protocol will be useful to researchers
interested in understanding the intricacies of T-cell maturation. It was
authored by Drs. Graham Anderson and Eric J. Jenkinson from the MRC Centre for
Immune Regulation at the

University of Birmingham (U.K.)
Other articles published today include methods for imaging neuronal activity
in zebrafish, examining gene expression patterns in fruit flies and frogs,
preparing DNA from mammals for genotyping, and identifying protein-protein
interactions in virtually any species. For a complete list of articles in the
August release of Cold
Spring Harbor Protocols
Production of endoribonuclease-prepared siRNA (esiRNA)
Frank Buchholz & Ralf Kittler
Here you find a general protocol to produce esiRNA in small scale. The protocol
is based on the in vitro digestion of long dsRNA with E. coli RNaseIII (an
enzyme that generates similar molecules as Dicer in eukaryotic cells). The
principle of the procedure is illustrated here.
1. Generation of PCR products for in vitro transcription of substrate RNAs
The template for in vitro transcription can be generated either by amplification
of cDNA inserts from clones using primers specific for the vector-backbone or
from cDNA preparations using target-specific primers appended with T7 promoter
When choosing cDNA clones or designing cDNA-specific vectors it should be taken
into account that the amplicon length is critical for the yield of dsRNA. We
observed highest yields with amplicon lengths between 400 and 1000 bp. While
shorter templates appear to require longer RNase III digestion times, we
occasionally observed annealing problems of the two ssRNAs when using templates
longer than 1000 bp.

Taq DNA polymerase and buffers (the dye in the BioTaq Red polymerase (Bioline)
is very useful to monitor transcription efficiency; see also in vitro
Forward and reverse primers with T7 promoter sequence 5’-CGTAATACGACTCACTATAGGG
added 5’ to a gene-specific or vector-backbone-specific primer sequence;
internal T7 promoter sites that are often present in vector backbones can be
utilized by choosing a primer to these sites.
Thermal cycler (e.g. MJ Research)
Template (e.g. clone or cDNA)

Experimental procedures
1. Mix the following reagents in a PCR tube:
5 µl 10 x PCR buffer
2 µl 50 mM MgCl2
4 µl 10 mM dNTP mix
1 µl 10 µM Forward primer
1 µl 10 µM Reverse primer
2 µl Taq DNA polymerase (1 U/µl)
1 µl template
PCR grade water to 50 µl
2. Amplify as follows: initial denaturation step of 94°C for 3 min; then 35
cycles of 94°C/30 sec, 60°C/30 sec and 72°C/30 sec; final elongation step of
72°C for 5 min.
3. Analyze products by standard agarose gel electrophoresis.
2. In vitro transcription and the production of dsRNA
PCR products can be used directly for in vitro transcription reactions without
purification. We routinely use 4 µl of the PCR product directly in in vitro
transcription reactions. When the BioTaq Red polymerase from Bioline was used,
the in vitro transcription reaction should result in a shift in color from red
to yellow.
The production of dsRNA including the synthesis of the two RNA strands and their
annealing is performed in a single tube. We have further streamlined this step
by skipping the conventional purification steps such as removal of DNA,
nucleotides and precipitation after in vitro transcription.

We routinely use the MEGAscript™ in vitro transcription kit (Ambion) to generate
RNA. Other suppliers should work accordingly.
Experimental procedures
1. Prepare a 10 µl reaction mix at room temperature as follows:

10 x transcription buffer 1.0 µl
75 mM ATP, GTP, CTP, and UTP 1.0 µl each (use 4 µl of "NTP mix")
PCR product 4.0 µl (~ 0.5 µg DNA template)
T7 RNA polymerase enzyme mix 1.0 µl
2. Incubate the reaction at 37°C overnight
3. Perform annealing in a thermal cycler as follows: 90°C for 3 min, ramp to
70°C with 0.1°C/sec, 70°C for 3 min, ramp to 50°C with 0.1°C/sec, 50°C for 3
min, ramp to 25°C with 0.1°C/sec. Do not freeze the long dsRNA as it tends to
form aggregates that hamper the downstream digestion.

3. Digestion and purification of dsRNA
Long dsRNA is partially digested to esiRNAs with a length of about 18-25 bp. The
subsequent purification effectively removes remaining DNA template,
unincorporated nucleotides and dsRNAs longer than 40 bp. We use the GST-RNaseIII
fusion protein as described in Yang et al. (http://www.pnas.org/cgi/content/full/99/15/9942).
The protein is easy to purify from E.coli and stable for a long time when stored
at -20°C. As E.coli RNaseIII overdigestion results in fragments sizes of ca.
12-15 bp we use a limited amount of the enzyme to achieve dsRNA length of 18-30
bp. It is therefore useful to perform a pilot serial dilution experiment with
each new batch of RNaseIII to determine the optimal amount of enzyme in the
digestion reaction.
Interestingly, the GST-part seems to shift the digestion products towards the
desired 21-30 bp range.
dsRNA digestion buffer, pH 7.9 (20 mM Tris-HCl, 0.5 mM EDTA, 5 mM MgCl2, 1 mM
DTT, 140 mM NaCl, 2.7 mM KCl, 5% (v/v) glycerol)
0.5 M EDTA, pH 8.0
4% (v/v) agarose in 1 x TBE electrophoresis buffer
25 bp DNA ladder (e. g., HyperLadder V from Bioline)
TE buffer, pH 7.9 (10 mM Tris-HCl, 1 mM EDTA)
70% (v/v) ethanol
Micro Bio-Spin Chromatography Columns (BioRad Laboratories)
Q Sepharose FastFlow (Amersham Biosciences)
Equilibration buffer, (20 mM Tris, 1 mM EDTA, 300 mM NaCl, pH 7.5)
Wash buffer, (20 mM Tris, 1 mM EDTA, 400 mM NaCl, pH 7.5)
Elution buffer, (20 mM Tris, 1 mM EDTA, 520 mM NaCl, pH 7.5)
Ethidium bromide, 10mg/ml (Sigma-Aldrich)
Orange G (Sigma-Aldrich); marker dye for esiRNAs
Experimental procedures
1. Mix 10 µl of the in vitro transcription reaction (∼25-50 µg dsRNA) with ∼4 µg
of GST-RNase III protein in 90 µl digestion buffer
2. Incubate at 23°C for 4 h on a thermomixer at 1200 rpm.
3. Run a 2-4 µl aliquot in 4% (v/v) agarose gel along with a 25 bp DNA ladder to
check the size range of digestion products. If the size range is not appropriate
(i. e., digestion products are too long) incubate for additional 2 h at 37°C and
check again. TIP: 4% agarose gels are a bit difficult to prepare. A good
solution is to heat the aragrose in TBE in a waterbath set to 95°C. It takes
some time to dissolve the agarose that way, but it avoids the generation of
4. Purify the digestion products immediately as follows, or terminate the
reaction by adding 4 µl 0.5 M EDTA.
5. Prepare spin columns for purification: Add 200 µl Q-Sepharose and 500 µl
equilibration buffer to the column. Spin at 1000 g for 1 min and discard the
6. Again, add 500 µl equilibration buffer, spin at 1000 g for 1 min and discard
the flow-through.
7. Load all of the digested dsRNA on the column and incubate for 5 min at room
8. Spin at 1000 g for 1 min and discard the flow-through.
9. Add 500 µl wash buffer, spin at 1000 g for 1 min and discard the
10. Add 300 µl elution buffer, spin at 1000 g for 1 min and collect the
11. Repeat step 10.
12. Add 500 µl isopropanol to the 600 µl eluted esiRNA and vortex. Store on ice
for at least for 30 min.
13. Spin at 16,000 g for 15 min at 4°C. Discard the supernatant, and wash the
pellet twice with cold 70% (v/v) ethanol.
14. Air-dry the esiRNA pellet for 10-15 min at room temperature and dissolve in
50 µl of 1 x TE buffer.
15. Run a 2-4 µl aliquot on a 4% (v/v) agarose gel, and measure OD260 in order
to quantify the esiRNA concentration (for practical reasons, we typically use
the same factor for quantification of esiRNA as for dsDNA).
About Researchers :
Applications to Cancer- and Stem Cell- Biology
Sequencing of whole genomes has provided new perspectives into the blueprints of
diverse organisms. Knowing the sequences, however, does not always tell us much
about the function of the genes that regulate development and homeostasis. Our
laboratory is using different strategies to dissect gene function in mammalian
cells relevant to cancer biology and stem cell research.
- use endoribonuclease prepared siRNA libraries
for functional genomic screens in mammalian cells
- study the role of runx1 in mouse development and leukaemogenesis.
- apply directed molecular evolution to design novel recombinases
for advanced genetic engineering.

selected publications.
Ralf Kittler, Gabi Putz, Laurence Pelletier, Ina Poser, Anne K. Heninger, David
Drechsel, Steffi Fischer, Irena Konstantinova, Bianka Habermann, Hannes Grabner,
Marie L. Yaspo, Heinz Himmelbauer, Bernd Korn, Karla Neugebauer, Maria T.
Pisabarro, Frank Buchholz: An endoribonuclease-prepared siRNA screen in human
cells identifies genes essential for cell division. Nature 432: 1036 -1040
Buchholz, F., Kittler, R., Slabicki, M., Theis,M., (2006).
Enzymatically prepared RNAi libraries.
Nature Methods, 3(9):696-700.
Riedel , C.G., Katis, V.L., Katou ,Y., Mori, S., Itoh, T., Helmhart, W., Galova,
M., Petronczki , M., Gregan, J., Cetin, B., Mudrak, I., Ogris, E., Mechtler, K.,
Pelletier, L., Buchholz , F., Shirahige, K., Nasmyth, K. (2006).
Protein phosphatase 2A protects centromeric sister chromatid cohesion during
meiosis I.
Nature, 441(7089):53-61.
Putz, G., Rosner, A., Nuesslein, I., Schmitz, N., and Buchholz, F. (2006).
AML1 deletion in adult mice causes splenomegaly and lymphomas.
Oncogene, 25(6):929-39.
Kittler, R., Heninger, A. K., Franke, K., Habermann, B., and Buchholz, F.
Production of endoribonuclease-prepared short interfering RNAs for gene
silencing in mammalian cells.
Nat Methods 2, 779-784
Max Planck Institute
of Molecular Cell Biology
and Genetics
Pfotenhauerstr. 108
01307 Dresden
phone +49 351 210-0
fax +49 351 210-2000
eMail info@mpi-cbg.de
MPI-CBG is part of the Max Planck Society. About 95% of Max Planck Society
expenditure is met by public funding from the Federal Government and the states.
The remaining 5% comes from donations, members contributions and from funded
projects. The 2002 budget is estimated to total €1,25 billion (2000: DM 2,34
MPI-CBG's budget covers the insitute's expenditure like personnel costs,
material costs, or co-operations with other institutions.
In The Images-

1.Optimization of dsRNA digestion. Lane1: dsRNA, no RNaseIII, Lane2: dsRNA, 1µg
RNaseIII, Lane 3: dsRNA, 2µg RNaseIII, Lane 4: dsRNA, 3µg RNaseIII, Lane 5: 4µg
RNaseIII. All reactions were incubated at 23°C for 4 hours and then loaded on a
4% agarose gel.

2.This figure shows 4 reactions after the in vitro transcription and annealing.
The second tube (failed) is still red and indicates that the in vitro
transcription did not work well. The third tube (good) shows a nice color shift
towards orange or yellow, indicative of an efficient reaction, most likely due
to a shift in pH

3.Schematic presentation of esiRNA production. Steps carried out to obtain a
pool of functional siRNAs are illustrated4.Dr. Frank Buchholz’s

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More Research

single stranded interference RNA(siRNA) though inserted is accesisble to rnase,,instead addition of histone coding genes segment into the existing dna ,,,results in additional foldings,resulting in silencing of genes.
Posted by: of hyderabad 28 January, 2009 00:48

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