Difference between revisions of "Antisense Oligo Knockdown in Mice"

Latest revision as of 20:43, 2 August 2011

Designing modified antisense oligonucleotide

• IDT website https://www.idtdna.com/Scitools/Applications/AntiSense/Antisense.aspx
• Insert nucleotide gene id (GI, written next to the NM_####)
• Ruleset - chimeric 20 mer -> calculate
• Check suggested sequences by blasting in blast->mouse nucleotide
• If using previously designed siRNA sequences make sure sequences are antisense! (direction on mRNA in blast should be from larger to smaller position). Regular siRNAs are duplexes of sense and antisense, so siRNA sequences used before are usually sense, which is the wrong sequence going from smaller to larger position.

modify sequence

1. first 5 and last 5 are mX (methylated RNA nucleotides)
2. all nucleotides are phosphorothiated (marked by * after the X)

Control oligo (routinely used in many papers) Original: CCUUCCCTGAAGGTTCCUCC Modified mC*mC*mU*mU*mC*C*C*T*G*A*A*G*G*T*T*mC*mC*mU*mC*mC

Ordering several small scale oligos to compare efficiency of knock down

(this step is possibly not necessary for siRNA sequences that have been used for knockdown in cell culture) Order from http://www.trilinkbiotech.com/products/oligo/quote.asp (can also be ordered from invitrogen)

• Details of small scale order:
• phosphorothiated backbone
  • sugar – other
  • scale: 0.2 uMole
  • purification: PAGE
• notes: * = phosphorothiated; mX = 2' O-methyl RNA bases
• transfect Hepa1-6 cells with 100nM oligo using Lipofectamine 2000:
• calculate final umoles of ASOs (OD/extinction coefficient)
• resuspend in umoleX10 ml (e.g. 0.37ml into 0.037 umole) to final concentration of 100uM
• transfect 1ul for each 1/12 wells, 0.5ul for each 1/24 well

Large scale ASO order

• order oligos with best knockdown large scale (approx $2000 for 100mg scale)
• http://www.trilinkbiotech.com/products/oligo/quote.asp
• Details of large scale order:
  • phosphorothiated backbone
  • sugar – other
  • scale: 100 mg
  • purification: anion exchange (AX) HPLC
• notes: * = phosphorothiated; mX = 2' O-methyl RNA bases
• arrives lyophilized.

Dilution of ASOs

• stock 100 mg in 1000 ul sterile normal saline
• aliqote to 40ul/100ul and store at -20.
• calculation of final concentration so that injection will be 5ul/gr mouse:

 Xug ASO/gr mouse
 5ul final conc./gr mouse
 Xug/5ul = X/5 mg/ml

Dose response pilot

• Inject IP 10mg/gr or 25mg/gr of control ASO/knockdown ASO twice a week for 2-3 weeks (treatment of more than 3 weeks causes liver toxicity).
• Test knockdown in liver and WAT by WB