Efficient construction of an inverted minimal H1 promoter driven siRNA expression cassette: facilitation of promoter and siRNA sequence exchange.
BACKGROUND
RNA interference (RNAi), mediated by small interfering RNA (siRNA), is an effective method used to silence gene expression at the post-transcriptional level. Upon introduction into target cells, siRNAs incorporate into the RNA-induced silencing complex (RISC). The antisense strand of the siRNA duplex then "guides" the RISC to the homologous mRNA, leading to target degradation and gene silencing. In recent years, various vector-based siRNA expression systems have been developed which utilize opposing polymerase III promoters to independently drive expression of the sense and antisense strands of the siRNA duplex from the same template.


RESULTS
We show here the use of a ligase chain reaction (LCR) to develop a new vector system called pInv-H1 in which a DNA sequence encoding a specific siRNA is placed between two inverted minimal human H1 promoters (approximately 100 bp each). Expression of functional siRNAs from this construct has led to efficient silencing of both reporter and endogenous genes. Furthermore, the inverted H1 promoter-siRNA expression cassette was used to generate a retrovirus vector capable of transducing and silencing expression of the targeted protein by>80% in target cells.


CONCLUSIONS
The unique design of this construct allows for the efficient exchange of siRNA sequences by the directional cloning of short oligonucleotides via asymmetric restriction sites. This provides a convenient way to test the functionality of different siRNA sequences. Delivery of the siRNA cassette by retroviral transduction suggests that a single copy of the siRNA expression cassette efficiently knocks down gene expression at the protein level. We note that this vector system can potentially be used to generate a random siRNA library. The flexibility of the ligase chain reaction suggests that additional control elements can easily be introduced into this siRNA expression cassette.
Nassanian
Hoorig
H
Department of Microbiology, Immunology and Molecular Genetics, David Geffen School of Medicine, University of California at Los Angeles, Los Angeles, California, United States of America.
Sanchez
Ana M
AM
Lo
Alice
A
Bradley
Kenneth A
KA
Lee
Benhur
B
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D020868:Gene Silencing; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D034741:RNA, Small Interfering / Q000235:genetics* / Q000378:metabolism; D034743:RNA-Induced Silencing Complex / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment
D034741:RNA, Small Interfering; D034743:RNA-Induced Silencing Complex
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