Article

[Recombinant adenovirus-mediated RNA silencing of tissue factor expression in human islet: an in vitro study].

Department of Kidney Transplantation, First Affiliated Hospital, School of Medicine, Xi'an Jiaotong University, Xi'an 710061, China.
Nan fang yi ke da xue xue bao = Journal of Southern Medical University 10/2007; 27(9):1299-302. pp.1299-302
Source: PubMed

ABSTRACT To construct a replication-incompetent recombinant adenovirus mediating short hairpin RNA (shRNA)-induced tissue factor gene silencing in the islet.
Four pairs of complementary oligonucleotides were designed and synthesized to create double-stranded oligonucleotides (ds oligo). The ds oligos were cloned into Pentr/U6 vector to construct the shuttle plasmid pENTR/U6-shRNA, which was transduced into human islets via liposome after sequence verification. The plasmid with the best silencing effect was identified by real-time RT-PCR, followed by homologous recombination with the adenovirus backbone plasmid. The functional clone was transfected into 293A cells to amplify the adenovirus, whose silencing effect against TF expression was tested using real-time RT-PCR and Western blotting.
The pENTR/U6-shRNA shuttle plasmid was constructed and verified by sequencing. The recombinant adenovirus-mediated shRNA against TF was constructed, and real-time RT-PCR and Western blotting demonstrated that the strongest silencing effect of the adenovirus against TF occurred on the 4th day following islet transfection.
Replication-incompetent recombinant adenovirus-mediated shRNA against TF has been successfully constructed, which has good silencing effect against TF expression in human islet in vitro.

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    Article: RNA interference for improving the outcome of islet transplantation.
    Feng Li, Ram I Mahato
    [show abstract] [hide abstract]
    ABSTRACT: Islet transplantation has the potential to cure type 1 diabetes. Despite recent therapeutic success, it is still not common because a large number of transplanted islets get damaged by multiple challenges including instant blood mediated inflammatory reaction, hypoxia/reperfusion injury, inflammatory cytokines, and immune rejection. RNA interference (RNAi) is a novel strategy to selectively degrade target mRNA. The use of RNAi technologies to downregulate the expression of harmful genes has the potential to improve the outcome of islet transplantation. The aim of this review is to gain a thorough understanding of biological obstacles to islet transplantation and discuss how to overcome these barriers using different RNAi technologies. This eventually will help improve islet survival and function post transplantation. Chemically synthesized small interferring RNA (siRNA), vector based short hairpin RNA (shRNA), and their critical design elements (such as sequences, promoters, and backbone) are discussed. The application of combinatorial RNAi in islet transplantation is also discussed. Last but not the least, several delivery strategies for enhanced gene silencing are discussed, including chemical modification of siRNA, complex formation, bioconjugation, and viral vectors.
    Advanced drug delivery reviews 12/2010; 63(1-2):47-68. · 11.96 Impact Factor
Data provided are for informational purposes only. Although carefully collected, accuracy cannot be guaranteed. The impact factor represents a rough estimation of the journal's impact factor and does not reflect the actual current impact factor. Publisher conditions are provided by RoMEO. Differing provisions from the publisher's actual policy or licence agreement may be applicable.

Keywords

4th day
 
adenovirus backbone plasmid
 
complementary oligonucleotides
 
double-stranded oligonucleotides
 
ds oligo
 
ds oligos
 
functional clone
 
homologous recombination
 
human islet
 
human islets
 
Pentr/U6 vector
 
pENTR/U6-shRNA shuttle plasmid
 
real-time RT-PCR
 
recombinant adenovirus-mediated shRNA
 
replication-incompetent recombinant adenovirus mediating short hairpin RNA
 
Replication-incompetent recombinant adenovirus-mediated shRNA
 
sequence verification
 
shRNA)-induced tissue factor gene
 
shuttle plasmid pENTR/U6-shRNA
 
TF expression
 

Zhao-lun Li