RNAi Hairpin Design for Zebrafish Transgenics
Jennifer H. Gutzman and Hazel Sive
Whitehead Institute for Biomedical Research and MIT
9 Cambridge Center
Cambridge, MA 02142
Background
shRNAs (short hairpin RNAs) expressed from a promoter are processed into interfering RNAs (RNAi) that can inhibit gene expression (Fig.1). Using a recently described procedure for zebrafish (Dong et al, 2009), the miR30 gene transcribed region can be used to efficiently produce RNA hairpins, where the normal miR30 hairpin is replaced with a hairpin targeting a gene of interest (GOI). Hairpins are then processed into interfering RNA.
Figure 1. Simple diagram of shRNA processing from a DNA vector with the miR30 backbone containing your shRNA of choice.
Green arrows indicate sense strand, red arrows indicate antisense strand.
Proceed as follows.
I. Design a hairpin to your GOI (see RNAi Hairpin Design). This will be part of a dsDNA molecule that will be inserted into the miR30 backbone.
II. Clone your GOI hairpin into the miR30 backbone. This step can be done either by
- ordering two complementary, purified oligos, anneal these and subclone into the cut vector. Such oligos run~$80 apiece,
for a total of ~$160 per test hairpin. - ordering unpurified oligos, for ~$30 each (total ~$60) anneal and purify these yourself to save on cost (see Oligo Purification Protocol).
III. Place this backbone behind a ubiquitous or a tissue-specific promoter. A GPF or RFP reporter can be transcribed
from the same promoter. It does not need to have an IRES if placed upstream of the miR backbone. Transgenesis can be
effected by standard meganuclease (I-SceI) or Tol2 methods. No protocol is included for this.
Reference: Dong, M., Fu, Y.F., Du, T.T., Jing, C.B., Fu, C.T., Chen, Y., Jin, Y., Deng, M., and Liu, T.X. (2009).
Heritable and lineage-specific gene knockdown in zebrafish embryo. PloS one 4, e6125.
I. RNAi hairpin Design
The protocol below describes design and cloning of oligos containing a hairpin, that will be inserted into a miR30 backbone.
1. Use the Invitrogen Block-IT RNAi designer website for finding RNAi sequences that would target your gene of interest.
2. Use this link and choose to design an shRNA, then enter your sequence or accession number.
3. Choose hits that are 4-5 stars and blast the sequences to the zebrafish genome to be sure your target gene is your only
hit (anything else should be looked at more carefully since off target effects are possible depending on your target sequence
and region of genome match within the hairpin).
4. Your RNAi target sequence should be a 21 mer.
5. Follow the example below to add in your sequence for designing your RNAi oligo. (should be a 71 mer)
6. YFP RNAi design example
7. Oligo design with color coding (YFP example, continued)
**NOTES:
All parts of the construct stay the same for each new targeting sequence EXCEPT FOR THE RED AND BLUE LETTERS. Be sure to properly create your 3' to 5' compliment.
When ordering, remember that the two oligos above, that you anneal are slightly different because of the NheI restriction site only on one side so you cannot have the program do an automatic reverse compliment.
II. Oligo Urea-PAGE Purification Protocol
1. Pour a large (~6inch X 6inch) denaturing UREA – PAGE gel.
% of your gel depends on the oligo size.
70 mer ~ 8-10%
using National diagnostics reagents.
No. EL-830
835 {order EL-833 (kit with all three)
840
The gel thickness depends on the amount you need to purify.
You will get a band at the correct size – then a smear of unpure oligo below your correct size band.
For single nucleotide resolution decrease your starting material and run the gel longer.
2. Resuspend lyophilized oligo in H2O
Add 100 ul H2O to 50nmol of oligo and resuspend oligo by mixing at room temperature.
take out about 50-20 ul oligo in H2O
add 2x loading dye
* Use loading dye containing both bromophenol blue and xylene cyanol with 8 M urea.
RECIPE for Urea Gel Loading Dye
2X mix is:
8M Urea (FW 60.06)
25mM EDTA (FW 372.2)
Use:
Urea: RSB gold capped molecular biology grade (cat. US 75826 1kg)
EDTA: Sigma molecular biology grade (cat. E-5134 500g)
Make:
1000mL, RNase-free (use no glass containers). Use only weigh boats from middle of stack and only plastic containers from Corning 0.22um filter apparatus. You will need 2 500mL filter apparatuses and 2 1000mL apparatuses (one to mix and one to actually filter).
A. Prepare 500mL, 500mM EDTA stock at pH 8.0 (see Maniatis 3 B.11)
a. to 93.1g EDTA, add 10g NaOH pellets; dump into 500mL filter bottle.
b. add nuclease-free H2O to 500mL
c. mix on orbital shaker until dissolved (overnight)
d. filter
2. Dissolve urea:
a. add 480g urea to 1000mL filter bottle
b. add nuclease-free H2O to 900mL
c. mix on orbital shaker until dissolved (overnight)
d. add 50mL, 500mM EDTA
e. top-off to 1000mL with nuclease-free H2O
f. filter
Preparing dyes to aliquot: we use 0.025% Xylene Cyanol and Bromophenol Blue, so weigh out 0.0125g per 50mL.
3. Load/Run gel with 0.5 x TBE running buffer
Start @ 2 watts 10-15 mins
Increase to 10-15 watts
Check the xylene dye front to determine how far to run your gel
Xylene runs at 55 bp with 8% gel
4. When finished take gel apart and put the gel between 2 pieces of saran wrap.
5. Look at the gel with UV shadowing over fluor-coated TLC plate.
Plates available from Applied Biosystems AM10110 10x10cm $41.80
Put saran wrapped gel on the plate – Quickly scan with UV light – Look for shadows. (The shadows are you bands and indicate the presence of DNA blocking the UV light)
Draw on the saran wrap around Shadow with Sharpie (do Not cut your gel on the Plate)
6. Cut out gel pieces and put the gel containing your oligo in an eppendorf tube.
Cut into pieces if necessary –
Want gel piece about ~1square centimeter (too big will lead to impure oligo!) You do not want to include any of the smear below your pure large band.
7. Add 400 ul 0.3M NaCl in H2O to tube (400 ul/tube of gel).
Rotate overnight @ 4°C.
DNA will come out of gel into salt!
8. Spin tube – acrylamide piece will be at bottom.
Collect Supernatant = oligo.
9. Add 2.5 x volumes of ethanol.
2.5 x volumes 400ul. (so 1 ml for 400 ul)
into -20°C at least 2 hrs.
10. Spin on high.
oligo = pellet! (10 min @4°C)
Dry pellet!
Resuspend in 50ul nuclease free water and spec for your actual DNA oligo concentration
11. Use for annealing / subcloning / ligations.
****NOTES:
Should recover ~ 25% of amount of oligo loaded on gel.
For example, based on starting material of ~ 660 ug loaded
Predict with 25% recovery = ~ 150 ug
Add 50 ul nuclease free water to each tube
Do spec reading
If see a range of 900 ng/ul – 2500 ng/ul, this means good recovery
Jennifer H. Gutzman and Hazel Sive
Whitehead Institute for Biomedical Research and MIT
9 Cambridge Center
Cambridge, MA 02142