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| Status |
Public on Dec 23, 2016 |
| Title |
Reconstitution of the RNAi response in human cells using drosophila gene products |
| Organism |
Homo sapiens |
| Experiment type |
Non-coding RNA profiling by high throughput sequencing
Expression profiling by high throughput sequencing
|
| Summary |
While mammalian somatic cells are incapable of mounting an effective RNA interference (RNAi) response to viral infections, plants and invertebrates are able to generate high levels of functional short interfering RNAs (siRNAs) of viral origin that can effectively control many infections. In Drosophila, the RNAi response is mediated by the Dicer 2 enzyme (dDcr2) acting in concert with two co-factors called Loqs-PD and R2D2. To examine whether a functional RNAi response could be reconstituted in human somatic cells by expression of these insect proteins, we expressed dDcr2, in the presence or absence of Loqs-PD and/or R2D2, in a previously described human cell line, NoDice/ΔPKR, that lacks functional forms of both the human Dicer (dcr) and EIF2AK2 (pkr) gene. Upon expression of dDcr2, Loqs-PD and R2D2 in these human cells, we observed the production of ~21-nt long siRNAs, derived from a co-transfected double stranded RNA (dsRNA) expression vector, that were loaded into the human RNA-induced silencing complex (RISC) and were able to significantly reduce the expression of a cognate indicator gene. We conclude that it is possible to at least partly rescue the ability of mammalian somatic cells to express functional siRNAs by using gene products of invertebrate origin.
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| Overall design |
Combinations of Drosophila Dicer2 and cofactors were transfected into 425-PKR cells to determine whether they were sufficient for genesis of siRNAs in mammalian cells.
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| Contributor(s) |
Kennedy EM, Kornepati AV, Bogerd HP, Cullen BR |
| Citation(s) |
27837013 |
| Submission date |
Jun 16, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Edward Matthew Kennedy |
| E-mail(s) |
edward.m.kennedy@gmail.com
|
| Phone |
9196845656
|
| Organization name |
Duke University
|
| Department |
Molecular Genetics and Microbiology
|
| Lab |
Byran Cullen
|
| Street address |
Department of Molecular Genetics and Microbiology, Box 3025
|
| City |
Durham |
| State/province |
NC |
| ZIP/Postal code |
27710 |
| Country |
USA |
| |
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| Platforms (1) |
| GPL11154 |
Illumina HiSeq 2000 (Homo sapiens) |
|
| Samples (12)
|
| GSM2202976 |
NoDice(4-25)-PKR Neg Deep |
| GSM2202977 |
NoDice(4-25)-PKR expressing N-terminally trunctated human Dicer N1 Deep |
| GSM2202978 |
NoDice(4-25)-PKR expressing dDcr2 Deep |
| GSM2202979 |
NoDice(4-25)-PKR expressing dDcr2 LoqsD Deep |
| GSM2202980 |
NoDice(4-25)-PKR expressing dDcr2 R2D2 Deep |
| GSM2202981 |
NoDice(4-25)-PKR expressing dDcr2 LoqsD R2D2 Deep |
| GSM2202982 |
NoDice(4-25)-PKR Neg Rip |
| GSM2202983 |
NoDice(4-25)-PKR expressing N-terminally trunctated human Dicer N1 Rip |
| GSM2202984 |
NoDice(4-25)-PKR expressing dDcr2 Rip |
| GSM2202985 |
NoDice(4-25)-PKR expressing dDcr2 LoqsD Rip |
| GSM2202986 |
NoDice(4-25)-PKR expressing dDcr2 R2D2 Rip |
| GSM2202987 |
NoDice(4-25)-PKR expressing dDcr2 LoqsD R2D2 Rip |
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| Relations |
| BioProject |
PRJNA325894 |
| SRA |
SRP076670 |
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