In utero electroporation and RNAi module
ATMN 2009
Joe LoTurco (Joseph.loturco@uconn.edu)
Brady Maher(Brady.Maher@uconn.edu)
Chris Fiondella(Christopher.Fiondella@uconn.edu)
Department of Physiology and Neurobiology
University of Connecticut, Storrs CT 06269
OVERVIEW
DAY 1 (July 6th)
Orientation Lecture- In vivo RNAi in mammalian CNS: Background and Experimental
Design (LoTurco)
shRNA construction
Orientation (LoTurco)
-Digest Plasmid
-Anneal shRNA oligos
-Gel purify digested Vector and annealed oligos
-Quick Ligation of annealed shRNA plasmid with gel purified vector.
-tansform bacteria
Electroporation
Orientation (LoTurco)
-Select and Mix plasmid combinations for electroporations
DAY 2. (July 7th)
Electroporation
Orientation: In utero and intraretinal electroporation: (intrauterine injections,
electroporation, tips and tricks) (Fiondella)
-Intrauterine surgery, injection and electroporation.
-Intraretinal pressure injection and electroporation
shRNA construction (cont’)
Orientation (Maher)
-PCR screen colonies for positive clones.
DAY 3 (July 8th)
In utero E-poration and Brain Slice Electrophysiology (Maher)
Electroporation (cont’)
-more surgeries
-Tamoxifen injections for inducible expression (optional)
shRNA construction (cont’)
-start mini prep shRNA constructs (optional).
DAY 4 (July 9th)
shRNA construction (cont’)
- mini prep shRNA constructs (optional).
electroporation (cont’)
-Harvest, fix and Image transfected retinas and cortex
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shRNA PLASMID CONSTRUCTION
mir30 Plasmid Vectors
There are three shRNA vectors you can choose from. Choose 1 or 2 out of three for your
constructions.
1) pCAG-mir30: Constitutive shRNA vector with mir30 sequence.
This plasmid is a vector that uses the Pol II CA promoter (beta actin promoter with the
CMV enhancer) to drive expression. It has miR30 sequence to allow the RNA to be
processed through the miRNA processing pathway. (Matsuda and Cepko 2007)
2) pCALSL-mir30: Conditional “on” floxed shRNA vector for conditional shRNA
expression.
This plasmid is similar to pCA-mir30 but has a loxp-neostop-loxp cassette upstream from
the miR30 sequence where you will insert the shRNA sequence. There are two pA
stretches in the floxed cassette for transcription termination. This vector allows for gated
expression of the shRNA following cre recombination of the loxp-neostop-loxp
sequence. (Matsuda and Cepko 2007)
3) pPBCA-mir30 : Constitutive miR30 shRNA vector with piggybac transposon TRs.
When co-transfected with a vector that expresses the Piggybac transposase (pCAPBASE) the transgene flanked by the 3 and 5' TRs (terminal repeats) will be integrated
into the genome of host cell. This is a non-viral system for integrating plasmid based
transgenes into the genome. This would be desirable for dividing cells or for situations
where stable transgene integration into a cell lineage is required. (unpublished Fuyi
Chen).
A) Digest vectors (30 l final Rx volume)
1) Prepare 1- 2 restriction digests.
For each Rx:
x l
2 g Plasmid DNA (stocks may be at different concentrations so dilute and pipette
accordingly)
3 l 10X NEB buffer (EcoR1 and Xho1 are both compatible in buffers 2,3, or 4)
1 l
EcoR1
1l
Xho1
y l H2O (Balance; 30-5-x)
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2) incubate at 37C for 1 hr.
3) Heat inactivate 65C for 20 minutes
B) Anneal shRNA Oligos. While the vector is digesting anneal oligos.
The oligos you will use to make the shRNA vectors have been predesigned according to a
set of criteria that have emerged over the past several years to increase the probability
that a given shRNA will create effective RNAi (see Appendix 2 for design considerations).
It is not 100% but using these critera result in approximately a 50% success rate. For
most human and mouse mRNAs shRNAs in lentiviral vectors have been made following
these design principles and are available from Open Biosystems.
1) -Resuspend each oligo in water to a final concentration of 0.25 nmols/ l. (eg. the
preps are 30 nmol so resuspend in 115 l H20. Vortex to fully re-suspend contents.
2) Anneal in 0.5 ml tube:
10 l oligo1 (positive strand)
10 l oligo 2 (negative strand)
2.5 l water
2.5 l 10x annealing buffer (1 M NaCl, 100 mM Tris pH 7.4)
3) Heat 200-300 mls of water to 90-95C and add tubes in the floating rack and let it all
cool slowly to room temp (1-2 hours). The cooling to room temp should be slow to
allow for efficient annealing. Briefly spin to recollect contents at the bottom of the tube.
Alternatively you can write a thermocycler program that goes from 95 C to 20 C over a
1.5 hour period.
C) Gel Purify digested vector.
1) Prepare and run a 0.8-1% gel.
2) Load 6 l of digest (or approximately 400 ng of linearized vector) + 1 ml of loading
buffer
3) run gel 100 V 20-30 minutes.
4) Check under UV that vector is cut completely (if not, then add more enzyme to
remaining digest and cut longer and run on gel)
5) Cut out and trim gel fragment containing the linearized vector
6) Purify fragment with Qiagen Gel Isolation Kit tm
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D) Ligate vector and annealed shRNA oligos.
1) Dilute annealed oligos 1:4000 in 0.5X annealing buffer.
2 μl of annealed oligos in
7.6 mls of H20
400 l of 10x annealing buffer ((1 M NaCl, 100 mM Tris pH 7.4)
The oligos are 65 bp ( approximately 100X smaller than the vector) and therefore to
achieve a molar ratio suitable for ligation the oligos must be highly diluted.
2) Add 1 l of diluted oligo to 50-100 ng of linearized gel purified vector (or
approximately 10 l from the gel isolation, C6)
3) Add 10 μl of 2X Quick Ligation Buffer and mix.
4) Add 1 μl of Quick T4 DNA Ligase and mix thoroughly.
5) Set up a separate reaction with 10 l of vector alone as a control.
6) Incubate at room temp for 10 minutes.
E) Transform chemically competent bacteria (Follow protocol on page 6 of Rusty’s
Lenti Protocol)
F) PCR screen colonies for insert-containing plasmids
1) Mark the underside of fresh ampicillin plates with a sharpie to divide into 5 or more
numbered segments.
2) Pick 4 colonies from each plate with a sterile pipette tip and streak each gently in a
segment of the marked amplicilin plate to make a master plate. Incubate 37 C overnight.
3) Transfer each pipette tip to 15 ul of water set up in numbered PCR tubes. Swish the tip
through the water and/or let the ejected pipette tip sit in the tube for 2-5 minutes.
4) Set up 6 25 ul PCR reactions for each transformation (4 with colonies, 1 positive
control with 2 ng of uncut plasmid vector; 1 l of 1:1000 dilution, and 1 negative control
water only control reaction.
15 l H20 (+bacteria 4 tubes, 2ng vector 1 tube, or water alone 1 tube)
10 l of master mix
master mix for 7 reactions:
7 l 10X Standard Reaction Buffer containing Mg2+
51 l water
3.5 l Deoxynucleatide mix (200 uM of each dNTP)
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3.5 l forward primer *
3.5 l reverse primer*
1.5 l taq polymerase
* There are two different sets of primers. You will use a particular set to identify inserts
according to the vector you chose.
Use Calslmir30for and Calslsmir30rev primers for pCA-LSL-mir30 construction
Use Camir30for and Camir30rev primers for both pPBCA-mir30 and pCA-mir30
5) Run Pcr
Thermocycle program:
1 cycle 94C- 5 minutes
35 cycles 94C-1minute, 58C-1 minute, 68C 1 minute.
1 cycle 72C- 10 minutes
Hold 4C
6) Run gel with 10 l of each PCR reaction + 2 l of 6 x loading gel.
7) Colonies that contain the appropriate insert should be approximately 400 bps while uninserted vector will run at approximately 330 bps
G (optional)
1) Start a miniprep culture for 2 positive clones containing inserted plasmid
2) Perform mini-prep.
3)** when you get home. If you want to confirm the sequence of the hairpin send DNA
for sequencing using either of the primers used to PCR screen the colonies. ** Hairpins
can be difficult to sequence and most sequencing facilities will have an optional set of
conditions for sequencing hairpins that will increase the chance of getting a good
sequence read.
IN VIVO ELECTROPORATION
In vivo eletroporation into cells of developing mammalian CNS (retina or cortex) permits
multiple plasmids to be co-transfected at a surprisingly high rate of co-transfection. In
three plasmid transfections with the same promoter approximately 90% of cells express
all three transfected plasmids. This allows for rather complex experimental designs by
simply choosing different combinations of plasmids. For example conditional shRNA
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plasmids can be combined with Cre expressing plasmids for conditional RNAi.
Alternatively, Cre expressing plasmids can be combined with conditional fluorescent
protein expressing plasmids for fate mapping. For the electroporation demonstrations
we have brought 10 different plasmids that can be used in different combinations for
conditional or constitutive expression. You have the option to combine just about any
combination of these in combinations of 2-3 plasmids.
A) Plasmid Mixture.
1) Select the plasmid combinations (2-3 plasmids in a mix) you wish to electroporate.
We should have enough animals for 2 postnatal electroporations and 3-4 fetal
electroporations for each group, so you can choose more that one combination. In fact,
you might consider designing a small experiment- possibly in collaboration with another
team. A description of the uses and features of the 10 plasmids and their maps are in
appendix 1 and appendix VI.
2) Approximately 1 l volumes can be injected into embryonic ventricles or neonatal
retina. A minimum 5-10 l volume mix is needed to load into the injection pipette. Each
plasmid in a mix should be at a final concentration of 0.5-1 g/l. In addition, a small
amount of fast green dye is added to the mix.
typical injection mix.
3 l 3 g/l plasmid 1
3 l 3 g/l plasmid 2
3 l 3 g/l plasmid 3 or
1 l 5% fast green in water
3) Mixes can be made the day before surgery and stored at 4C.
B) In utero electroporation
1) Pull and fill glass injection pipettes. Cut the pulled pipettes 1 cM from the taper with a
forceps. We find that this allows for a sharp enough pipette that is also rigid enough to
inject into the sclera or through the uterus. Just prior to surgery load plasmid injection
mix into pulled glass injection pipettes. Load the injection pipette into the pipette holder
of the pressure injector and secure by gently tightening.
anesthesia:
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2) For in utero electroporation induce anesthesia with ip injetion of ketamine/xylazine
(100/7 mgs/Kg), It is often helpful to pre-anesthetize the animal first with isoflurane.
Additional 1/4 doses of xylazine/ketamine are given as needed to pregnant dams.
3) For intraretinal injections pups (0-5 days) can be iced on wet ice for 3-5 minutes or
until they stop moving. Retinal injections are made in minutes and pups can then be
revived by warming.
4) After induction of a deep anesthetic state with ketamine and xylazine the animals for
in utero electroporation are prepped for incision by shaving and disinfecting with
betadine around abdominal incision site. Prior to incision inject the animals with a 0.1
mg/kg s.c (subcutaneous) dose of metacam analgesic
5) Cut a 4-5 CM midline incision through skin and then abdominal muscle to expose
uterine horns.
6) Drape the incision site with sterile draping with a hole cut where the incision is made.
7) Wet the draping with preheated sterile saline + penstrep 37C.
8) Gently pull out embryos within the uterus 4-5 at a time onto the sterile and irrigated
drape. Intermittently wet embryos and uterus with saline to prevent drying. Count the
number of embryos in each uterine horn and draw a notebook map numbering each
embryo to keep track of injections. Each embryo can serve as a separate experiment as
they will not shift position within the uterine horns following surgery.
9) For injection, gently place a spatula under the head of the embryo. Illuminate the
embryos well with a fiber optic to visualize the head and skull sutures.
10) Position the head by manipulating embryo with both the spatula and free fingers until
the head of the embryo is pushed up directly against the uterine wall and held in place
with left hand (right hand if you're left handed). Positioning the embryo is a critical part
and the part that takes the most practice. Too much finger pressure can damage the
embryonic membranes leading to lethality and not enough will make injection difficult.
11) Using the blood vessels running along the skull suture as a guide inject pipette
through the uterus, and into the lateral ventricles of the embryo.
13) Pressure inject by depressing the foot pedal controller. (7-15 psi of pressure for .1
seconds should be sufficient. If not, inject again and/or increase the pressure or injection
time). After injection you will see the ventricle fill with fast green. Keep the pipette in
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for a few seconds after injection and then withdraw. Keep the embryo held gently against
the uterous for a few more seconds.
14) To electroporate wet the embryo with saline and position the electrode paddle so the
positive electrode is adjacent to the hemisphere that was injected. The negative paddle
should be on the side of the head opposite to the injection.
15) Pulse once 80V for 50 msec. repeat for each embryo. It is also possible to make a
second injection and electroporation into the opposite hemisphere for the same embryo,
but this can reduce survival rates.
16) After all or most embryos are electroporated return all to the body cavity and fill
cavity with warm sterile saline with pen-strep.
17) Close muscle and then skin with separate strings of suture. 4.0 sterile ethicon or
similar suture.
18) Return animal to cage on its back on a heating pad for recovery (1-2 hours). Return
animal to the colony after it is fully ambulatory and moving normally. The next day give
animal another injection of metacam analgesic (0.1 mg/kg) or sacrifice and harvet
embryos.
C) Intraretinal electroporation
1) Injection pipettes are prepared the same as above.
2) Remove all pups from the mother. It is best to do the same procedures to all pups and
return them at the same time, otherwise mothers often separate out the manipulated pups
and kill them.
3) For intraretinal injections pups (0-5 days) can be iced on wet ice for 3-5 minutes or
until they stop moving. Retinal injections are made in minutes and pups can then be
revived by warming after electroporation.
4) To expose the eye make a 5 mm incision with a scissors through the eyelid along the
line that marks the fusion of the upper and lower eyelids. Avoid cutting the cornea by
lifting eyelid with a forceps.
5) While holding the head and eye firmly with finger tips, insert injection pipette into the
eye at the margin of the retina and cornea. The margin of the retina is clearly visible as
an opacity.
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6) Once inserted Position the injection pipette so that it is roughly tangential to the
curvature of the eye and move the tip to the inner surface of the sclera. The target for the
injection is the small space between the pigment epithelia and the retina along the inner
wall of the eye. Injections into the vitreous will not result in very good electroporation.
7) Pressure inject until a crescent of blue is seen along the wall of the eye. It may be
necessary to slightly adjust the position of the pipette tip, and to increase the amount of
pressure or time of injection.
8) Inject the other retina.
9) To electroporate position the paddles so they cover each eye and electroporate (100 V
60 msec). Reverse the polaritiy of the paddles and electroporate again.
10) If electroporation does not cauterize the eyelids closed then use a small amount of
super glue to close the incision.
11) Warm the pups in hands or in 37C water bath until they are pink and start breathing.
12) Collect pups in a separate cage or container and return pups back to the mother as a
single group.
D) Tamoxifen injections (optional)
If you chose the pCAG ErCreEr plasmid then you will want to activate the Cre by
injection of tamoxifen. This should be done at least one day after electroporation and one
day before harvest.
1) The stock tamoxifen is 20 mg/ml in ETOH
2) Dilute to 2 mg/ml in vegetable oil. Load into a 1CC syringe.
3) Inject .3-.4 mgs total i.p (150-200 l).
E) Harvest and image tissue.
1) Overdose adult animals with isoflurane or ketamine and xylazine until respiration
ceases.
2) Pups can be killed by anesthetizing with ice or isoflurane and then decapitating.
3) Collect embryo heads or neonatal eyes on ice in PBS, HBSS, or Saline.
4) Dissect forebrains from skulls with forceps. Divide the two hemispheres, and remove
as much of the meninges as possible.
5) Dissect retina from eyes by cutting with a small scissors either around the cornealretinal margin and gently teasing out the retina (it is a pearly white sheet that easily
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separates from the eye) or by cutting from the point the optic nerve head exits the eye to
open the eye.
5) Fix the dissected forebrains and retinas in 4 % parafomraldehyde in PBS at 4 C for
0.5-1 hr. Rinse in Cold PBS. It is best to work with paraformaldehyde solutions in the
chemical fume hoods.
6) Check whole mounts for fluorescence patches of transfection
7) The fluorescent signals for mRFP, DsRed or GFP, can be imaged easily in whole
mounts of retina or cortex and with confocal it may be possible to attain some level of
cellular resolution. If you want to image at greater detail you will need to vibratome
section the positive tissue.
8) For vibratome sectioning, embed the fluorescently labeled forebrain or retina in 2%
agarose in PBS. To embed tissue pour molten agarose /PBS into cell culture wells or 10
cm bacterial plates. When the agarose is still warm add tissue. Block agarose and
section on vibratome at 80-100 microns.
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APPENDIX 1
PLASMIDS (maps in Appendix VI)
shRNA miR30 plasmid vector.
pCAG-mir30
pCALSL-mir30
pPBCA-mir30
SELECT FROM THESE FOR EPORATION
For constituitive FP expression
pCAG-eGFP: Constituitive expression of eGFP under a strong promoter ubiquitously
active
pCAG-mRFP: Constituitive expression of mRFP under a strong promoter.
For conditional FP expression gated "on" by Cre recombinase expression
pCALNL-eGFP
pCALNL-DsRED: the lox-neo stop must be removed by recombination prior to
expression of either eGFP or DsRed
For Cre expression by different promoters
pNestin-Cre : Neural progenitor promoter driving Cre
pGFAP-Cre : astrocyte and neural progenitor promoter
pCAG-Cre: ubiquitous promoter driving Cre
For expression of Er-Cre-Er under the control a ubiquitously active promoter.
pCAG-ErCreEr: ErCreEr will only induce Cre recombination in the presence of
tamoxifen which allows for the protein to enter the nucleus.
For lineage labeling and non-viral genomic integration of plasmid based transgene
pCAG-PBASE: The ubiquitous CA promoter driving expression of piggybac transposase.
This plasmid should be conmbined with pZGS for non-viral integration of transgenes in
pZGS.
p ZGS: The insert is flanked by piggybac TRs. The insert contains a splice acceptor
followed by beta-galactosidase followed by the CA promoter uprstream from eGFP. The
splice aceptor is for exon trapping. The entire insert inside the TRs has Lox-p sites so the
vector can also be used as a conditional GFP "OFF" by Cre recombinase.
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APPENDIX 2
shRNA Design considerations
If you are making shRNA vectors for mouse or human consider using the premade
sequences selected by the RNAi consortium.
http://www.broadinstitute.org/rnai/trc
There is a searchable database of produced shRNA vectors that have been placed in
Lenitviral vectors with an internal U6 promoter. These plasmids are available from open
biosytems for approximately $200. For making specialized vectors selecting from these
defined target sequences is a good approach.
Searchable database:
http://www.broadinstitute.org/genome_bio/trc/publicSearchForHairpinsForm.php
http://www.broadinstitute.org/genome_bio/trc/publicGetTransFromGeneNameForm.html
For designing your own shRNA sequences it is best to follow the design algorithm
consideration established by Broad. The most important are ** below.
The siRNA Target designer (http://www.promega.com/siRNADesigner/program/) does a
nice job at finding many potential sequence targets following rules 2,3, 4 and 5. Simply
paste in your mRNA sequence target and it will return many potential sequences that you
can screen through.
TRC (The RNAi Consortium) Algorithm Rules
1) Avoid SNPs
**2) THREE PRIME CLAMP.
Give precedence to candidates with weaker base-pairing at the 3 prime end
of the putative candidate; reward 5 if last three positions are A or T, 4.5 if
last two are A|T and third from is G|C and fourth is A|T; 4 if the last two are A|T;
2 if the last base is A|T; penalty is .2 if last two positions are G|C; .5 if the last
base is G|C; 0.8 if the last base is G|C and previous two are A|T.
**3) FIVE PRIME CLAMP.
Give precedence to a candidates with stronger base-pairing (GC) at the 5
prime end of the putative candidate, referred to as five_prime_clamp;
penalty/reward .01 if first two positions are GG, .0001 if first two are TT; 2.5 if first
four are (G|C){4}; 2.4 if first three positions are G|C{3}; 2.2 if begins
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(CC|CG|GC)(A|T)(G|C); 2 if begins (CC|CG|GC); 2 if begins (GC); 1.25 if begins
(G|C); 1 if begins (A|T)(G|C); .5 if begins ((A|T){2}
**4) GC content
gcContent: extremes of GC percentage are penalized; candidates with GC <
30% are penalized .01; with > 70% the penalty is .01; with GC between 30-50%
the candidate gets a reward of 3; with GC >60 and <70% the reward/penalty is
1
5) Avoid-four-in-a-row
6) Avoid seven (G|C) in a row
7) Internal loop penalty
penalize candidates that can form a AAABBB loop with a 0.7 penalty
8) internal AT skew
We want to reward moderately AT rich regions from 7 through 10; if all four are
A|T, rewards is 2.2; if 3 of 4 are A|T, the reward is 2, if 2 of 4 is A|T, the reward is
1.5; if 1 or 4 is A|T, the penalty is .7; if none of the four are A|T, the penalty is 0.5
9) internal AT flanking skew
we want to reward moderately AT-rich sequences at position 6 and 11; if both are
AT, the reward is 1.2; if 1 is either A|T, the reward is 1 and if neither is A|T, the
penalty is 0.85
10) penalize AA start
Blast sequences against the genome or refseq database for the organism that
you are using to make sure that no other mRNA is a match in more than 16
bases.
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APPENDIX 3
shRNA oligos for mir30 based shRNA vectors.
Mir 30 loop sequence
tagtgaagccacagatgta
Xho1 5’overhang
TCGA
EcoR1 5’overhang
AATT
Christy Welshhans
Igf2bp1 (rat) (ENSRNOG00000006122) NM_175594
No mouse broad clones
http://www.promega.com/siRNADesigner/program/
examples:
GGG TGC CAC CAT CCG AAA CAT
GCC AGC GCC TCC ATC AAG ATT
GCG GCC AGT TCT TGG TCA AAT
GCC CTG AAG GTT TCC TAC ATA
EcoR1
Sense Sequence
miR30 loop
Anti-sense
5’ TCGA GCC CTG AAG GTT TCC TAC ATA tagtgaagccacagatgta TAT GTA GGA AAC CTT CAG GGC 3’
3’ CGG GAC TTC CAA AGG ATG TAT atcacttcggtgtctacat ATA CAT CCT TTG GAA GTC CCG TTAA 5’
Xho1
Primers 5’-3’:
5’ TCGA GCC CTG AAG GTT TCC TAC ATA TAGTGAAGCCACAGATGTA TAT GTA GGA AAC CTT CAG GGC 3’
5’ AATT GCC CTG AAG GTT TCC TAC ATA TACATCTGTGGCTTCACTA TAT GTA GGA AAC CTT CAG GGC 3’
Henrik Boije
FoxN4 (Gallus gallus) NM_001083359 XR_027142
Gallus gallus forkhead box N4 (FOXN4),
http://www.promega.com/siRNADesigner/program/
GGA GCA CCT GGA GCA ATG ATT
GTG ACT TGC AGT CCT TGT CAT
GCC ATA GCC CAG AAT ACA CAA
GCA ATC TAC AAC ACA TCG TAT
EcoR1
Sense Sequence
miR30 loop
Anti-sense
5’ TCGA GCA ATC TAC AAC ACA TCG TAT tagtgaagccacagatgta ATA CGA TGT GTT GTA GAT TGC 3’
3’ CGT TAG ATG TTG TGT AGC ATA atcacttcggtgtctacat TAT GCT ACA CAA CAT CTA ACG TTAA 5’
Xho1
Primers 5’-3’:
5’ TCGA GCA ATC TAC AAC ACA TCG TAT TAGTGAAGCCACAGATGTA ATA CGA TGT GTT GTA GAT TGC 3’
5’ AATT GCA ATC TAC AAC ACA TCG TAT TACATCTGTGGCTTCACTA ATA CGA TGT GTT GTA GAT TGC 3’
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Joe Phillips
REST (RE-1) mouse ENSMUST00000113448
Broad sequence targets
CGC GGC TTC TAA GAA GTG TAA
CCA GAA ATA TAC AGC GCC AAT
CGC CCG TAT AAA TGT GAA CTT
CCC AAG ACA AAG ACA AGT AAA
CCG CTG TGG ATA CAA TAC CAA
EcoR1
Sense Sequence
miR30 loop
Anti-sense
5’ TCGA CGC GGC TTC TAA GAA GTG TAA tagtgaagccacagatgta TTA CAC TTC TTA GAA GCC GCG 3’
3’ GCG CCG AAG ATT CTT CAC ATT atcacttcggtgtctacat AAT GTG AAG AAT CTT CGG CGC TTAA 5’
Xho1
Primers 5’-3’:
5’ TCGA CGC GGC TTC TAA GAA GTG TAA TAGTGAAGCCACAGATGTA TTA CAC TTC TTA GAA GCC GCG 3’
5’ AATT CGC GGC TTC TAA GAA GTG TAA TACATCTGTGGCTTCACTA TTA CAC TTC TTA GAA GCC GCG 3’
Joe Biedenkapp
Dlgap1 (mouse) (ENSMUSG00000003279)
NM_177639
Broad sequences:
CCG GAG TAA CAA TGA CAT CAA
CGA GTC GCA AAC TCT GAG ATA
GCA GTT GTC CAT AGA GAA TAT
CGA CCT GGA CTT CCA TGA TAA
GCC CGA GAG GTT TAT CAG AAA
GCC CGA GAG GTT TAT CAG AAA
EcoR1
Sense Sequence
miR30 loop
Anti-sense
5’ TCGA GCC CGA GAG GTT TAT CAG AAA tagtgaagccacagatgta TTT CTG ATA AAC CTC TCG GGC 3’
3’ CGG GCT CTC CAA ATA GAC TTT atcacttcggtgtctacat AAA GAC TAT TTG GAG AGC CCG TTAA 5’
Xho1
Primers 5’-3’:
5’ TCGA GCC CGA GAG GTT TAT CAG AAA TAGTGAAGCCACAGATGTA TTT CTG ATA AAC CTC TCG GGC 3’
5’ AATT GCC CGA GAG GTT TAT CAG AAA TACATCTGTGGCTTCACTA TTT CTG ATA AAC CTC TCG GGC 3’
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Jean Abel
Calb1 (mouse) (ENSMUSG00000028222)
Broad sequences.
GCC AAC AAT AAA TCC
GCA ACG GAT ACA TAG
GAT TGG AGC TAT CAC
CTC ATG CTG AAA CTA
GAC CTC ATG CTG AAA
TAG
ATG
CGG
TTT
CTA
AAT
AAA
AAA
GAT
TTT
GCC AAC AAT AAA TCC TAG AAT
EcoR1
Sense Sequence
miR30 loop
Anti-sense
5’ TCGA GCC AAC AAT AAA TCC TAG AAT tagtgaagccacagatgta ATT CTA GGA TTT ATT GTT GGC 3’
3’ CGG TTG TTA TTT AGG ATC TTA atcacttcggtgtctacat TAA GAT CCT AAA TAA CAA CCG TTAA 5’
Xho1
Primers 5’-3’:
5’ TCGA GCC AAC AAT AAA TCC TAG AAT TAGTGAAGCCACAGATGTA ATT CTA GGA TTT ATT GTT GGC 3’
5’ AATT GCC AAC AAT AAA TCC TAG AAT TACATCTGTGGCTTCACTA ATT CTA GGA TTT ATT GTT GGC 3’
Michael Steiss
Transgelin (rat)
NM_031549
http://www.promega.com/siRNADesigner/program/
GCT
GCT
GGC
GCA
GAA
AGT
TAG
CGT
GAA
GGA
TGG
CAT
TGG
GTG
AGT
TGG
CGT
GAT
GGA
CCT
GAT
TGT
TTG
TCA
TCT
AAT
TAA
AAT
EcoR1
Sense Sequence
miR30 loop
Anti-sense
5’ TCGA GCA CGT CAT TGG CCT TCA AAT tagtgaagccacagatgta ATT TGA AGG CCA ATG ACG TGC 3’
3’ CGT GCA GTA ACC GGA AGT TTA atcacttcggtgtctacat TAA ACT TCC GGT TAC TGA ACG TTAA 5’
Xho1
Primers 5’-3’:
5’ TCGA GCA CGT CAT TGG CCT TCA AAT TAGTGAAGCCACAGATGTA ATT TGA AGG CCA ATG ACG TGC 3’
5’ AATT GCA CGT CAT TGG CCT TCA AAT TACATCTGTGGCTTCACTA ATT TGA AGG CCA ATG ACG TGC 3’
17
Sundra Chetty
Olig1 (rat)
NM_021770
http://www.promega.com/siRNADesigner/program/
GCG TCC CTT CTC CCT AAA CCT
GGA CGC GCT GCG CGA AGT TAT
GCA GCT GAG GCG CAA GAT CAA
GCT GCT CGC CCG CAA CTA CAT
GCG CTG CGC GAA GTT ATC CTA
EcoR1
Sense Sequence
miR30 loop
Anti-sense
5’ TCGA GCA GCT GAG GCG CAA GAT CAA tagtgaagccacagatgta TTG ATC TTG CGC CTC AGC TGC 3’
3’ CGT CGA CTC CGC GTT CTA GTT atcacttcggtgtctacat AAC TAG AAC GCG GAG TCG ACG TTAA 5’
Xho1
Primers 5’-3’:
5’ TCGA GCA GCT GAG GCG CAA GAT CAA TAGTGAAGCCACAGATGTA TTG ATC TTG CGC CTC AGC TGC 3’
5’ AATT GCA GCT GAG GCG CAA GAT CAA TACATCTGTGGCTTCACTA TTG ATC TTG CGC CTC AGC TGC 3’
Brady Maher
Cx 36 Gjd2 (Gja9) (rat)
NM_019281
http://www.promega.com/siRNADesigner/program/
GTG GGA GCA AGC GAG AAG ATA
GAC GGT CTT TCT GGT GTT CAT
GCC AAG AGG AAG TCA GTC TAT
GTG GGA GCA AGC GAG AAG ATA
EcoR1
Sense Sequence
miR30 loop
Anti-sense
5’ TCGA GTG GGA GCA AGC GAG AAG ATA tagtgaagccacagatgta TAT CTT CTC GCT TGC TCC CAC 3’
3’ CAC CCT CGT TCG CTC TTC TAT atcacttcggtgtctacat ATA GAA GTG CGA ACG AGG GTG TTAA 5’
Xho1
Primers 5’-3’:
5’ TCGA GTG GGA GCA AGC GAG AAG ATA tagtgaagccacagatgta TAT CTT CTC GCT TGC TCC CAC 3’
5’ AATT GTG GGA GCA AGC GAG AAG ATA tacatctgtggcttcacta TAT CTT CTC GCT TGC TCC CAC 3’
18
APPENDIX 4
GLOSSARY of RNAi terms
Adapted from http://www.rnaiweb.com/RNAi/RNAi_Glossary/
Argonaute - A family of proteins containing multiple domains and involved in RNA interference
(RNAi). Argonatue is the main component of RNAi effector complex, the so called siRNA
induced silencing complex (RISC) and aids in target recognition and cleavage during
RNAi.
Dicer - Dicer is a member of the RNase III family of nucleases that specifically cleave doublestranded RNAs. Dicer processes long dsRNA into siRNA of 21-23 nt.
Interferon - A small and highly potent molecule that functions in an autocrine and paracrine
manner, and that induces cells to resist viral replication. This term is related to RNAi
because in mammals introduction of dsRNA longer than 30 nt induces a sequencenonspecific interferon response.
MicroRNA (miRNA) - Micro-RNAs (miRNA) are single-stranded RNAs of 20-22-nt that are
processed from ~70-nt hairpin RNA precursors by Rnase III nuclease Dicer. Similar to
siRNAs, miRNAs can silence gene activity via destruction of homologous mRNA in plants
or blocking its translation in plants and animals.
Post-Transcriptional Gene Silencing - Post-transcriptional gene silencing (PTGS) is a
sequence-specific RNA degradation system designed to act as an anti-viral defense
mechanism. A form of PTGS triggered by transgenic DNA, called co-suppression, was
initially described in plants and a related phenomenon, termed quelling, was later
observed in the filamentous fungus Neurospora crassa
Ribozyme - Ribozymes are RNA molecules that act as enzymes in the absence of proteins.
RNA-Directed DNA Methylation - RNA-directed DNA methylation (RdDM) is an RNA directed
silencing mechanism found in plants. Similar to RNA interference (RNAi), RdDM requires
a double-strand RNA that is cut into short 21-26-nt fragments. DNA sequences
homologous to these short RNAs are then methylated and silenced.
RNA-Induced Silencing Complex - RNA-induced silencing complex (RISC) is an siRNAdirected endonuclease, catalyzing cleavage of a single phosphodiester bond on the RNA
target.
RNAi - RNA Interference (RNAi), a term coined by Fire et al in 1998, is a phenomenon that
small double-stranded RNA (referred as small interference RNA or siRNA) can induce
efficient sequence-specific silence of gene expression.
RNAi Trigger - RNAi triggers are double-stranded RNAs containing 21-23 nt sense and
antisens strands hybridized to have 2 nt overhangs at both 3' ends.
shRNA - shRNA or short hairpin RNA is an RNA molecule that contains a fragment of a sense
strand and an antisense strand, and a short loop sequence between the sense and
antisense fragment. Due to the complementarity of the sense and antisense fragments in
their sequence, such RNA molecules tend to form hairpin-shaped double-stranded RNA
(dsRNA) by flipping back on the loop sequence. Usually, shRNA is cloned into a vector
so that once the vector is introduced into a cell, the shRNA can be transcribed by a pol III
type promoter in the vector. The expressed shRNA is then exported into the cytoplasm
where it is processed by dicer into siRNA which then triggers RNAi.
Small Interfering RNA (siRNA) - Small Interfering RNA (siRNA) is 21~23-nt double-stranded
RNA molecules. It guides the cleavage and degradation of its cognate RNA.
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APPENDIX VI Plasmid Maps
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