Comparison of chicken 7SK and U6 RNA polymerase III promoters for short hairpin RNA expression.
BACKGROUND
RNA polymerase III (pol III) type 3 promoters such as U6 or 7SK are commonly used to express short-hairpin RNA (shRNA) effectors for RNA interference (RNAi). To extend the use of RNAi for studies of development using the chicken as a model system, we have developed a system for expressing shRNAs using the chicken 7SK (ch7SK) promoter.


RESULTS
We identified and characterised the ch7SK promoter sequence upstream of the full-length 7SK small nuclear RNA (snRNA) sequence in the chicken genome and used this to construct vectors to express shRNAs targeting enhanced green fluorescent protein (EGFP). We transfected chicken DF-1 cells with these constructs and found that anti-EGFP-shRNAs (shEGFP) expressed from the ch7SK promoter could induce efficient knockdown of EGFP expression. We further compared the efficiency of ch7SK-directed knockdown to that of chicken U6 (cU6) promoters and found that the efficiency of the ch7SK promoter was not greater than, but comparable to the efficiency of cU6 promoters.


CONCLUSIONS
In this study we have demonstrated that the ch7SK promoter can express shRNAs capable of mediating efficient RNAi in a chicken cell line. However, our finding that RNAi driven by the ch7SK promoter is not more efficient than cU6 promoters contrasts previous comparisons of mammalian U6 and 7SK promoters. Since the ch7SK promoter is the first non-mammalian vertebrate 7SK promoter to be characterised, this finding may be helpful in understanding the divergence of pol III promoter activities between mammalian and non-mammalian vertebrates. This aside, our results clearly indicate that the ch7SK promoter is an efficient alternative to U6-based shRNA expression systems for inducing efficient RNAi activity in chicken cells.
Bannister
Stephanie C
SC
CSIRO Livestock Industries, Australian Animal Health Laboratory, Geelong 3220, Australia. stephanie.bannister@csiro.au
Wise
Terry G
TG
Cahill
David M
DM
Doran
Timothy J
TJ
D000818:Animals; D045744:Cell Line, Tumor; D002645:Chickens; D015870:Gene Expression; D018390:Gene Targeting; D017930:Genes, Reporter; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D009690:Nucleic Acid Conformation; D011401:Promoter Regions, Genetic / Q000235:genetics*; D034622:RNA Interference; D012320:RNA Polymerase III / Q000096:biosynthesis / Q000235:genetics*; D034741:RNA, Small Interfering / Q000096:biosynthesis*; D012342:RNA, Small Nuclear / Q000235:genetics*
D034741:RNA, Small Interfering; D012342:RNA, Small Nuclear; C405486:U6 small nuclear RNA; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D012320:RNA Polymerase III