Target region
|
Target sequence
|
Nucleotide position
|
CCR5 reduction on MAGI-CCR5
|
---|
CCR5-1
|
aagtgtcaagtccaatctatgac
|
13
|
+++
|
CCR5-2
|
aagagcatgactgacatctacct
|
186
|
++
|
CCR5-3
|
ctgacaatcgataggtacctggc
|
366
|
-
|
CCR5-4
|
gtgacaagtgtgatcacttgggt
|
442
|
-
|
CCR5-5
|
ttgtcatggtcatctgctactgg
|
624
|
++
|
CCR5-6
|
cagtagctctaacaggttggaca
|
809
|
+
|
CCR5-7
|
aaggtcttcattacacctgcagc
|
517
|
+/-
|
CCR5-8
|
aagttcagaaactacctcttagt
|
909
|
+++
|
- Eight shRNAs against human CCR5 were selected by a published algorithm[7]. shRNA target sequences are shown in the second column. Corresponding nucleotide positions of the target sequence in CCR5 mRNA are shown in the 3rd column. MAGI-CCR5 cells were transduced with lentiviral vectors expressing shRNA under the U6 promoter. To determine the expression levels of CCR5 on cell surface, the cells were cultured for 4 days and stained with APC conjugated anti-human CCR5 monoclonal antibody. The reduction levels of in EGFP+ population was analyzed by flow cytometry and shown as following criteria (+++ more than 10 fold reduction, ++ 10 fold reduction, + 3–5 fold reduction, +/- 2 fold reduction, – no reduction).