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LAS NORMAS GENERALES DE CONSTRUCCIÓN PARA EL SECTOR DE NUEVOS DESARROLLOS , serán las siguientes:
5.2.3.1 Preparation of DNA of esiRNA Region
The pre-‐cloned full-‐length DUBs plasmids and the esiRNA region-‐containing plasmids essentially represent two different sources of esiRNA region. For the former, esiRNA regions were PCR-‐amplified from the plasmids with corresponding primers (Fig 5.6). Occasionally, troubleshooting needs to be done to optimize PCR conditions, including annealing temperature, primer concentration and plasmid concentration. PCR products were purified from gel after DNA agarose gel electrophoresis using Gel Extraction Kit from QIAGEN according to the manufacturer’s instruction.
Figure 5.7. PCR amplification of esiRNA region from precloned plasmids. High fidelity DNA polymerase, Pfu was used for PCR reaction and reactions were set up in duplicates. Samples were analysed by DNA agarose gel electrophoresis.
On the other hand, the esiRNA-‐region containing plasmids were multiplied by growing the transformed bacteria in culture medium and the plasmids were subsequently purified using the QIAGEN Plasmid MiniPrep Kit. These plasmids cannot be used immediately for in vitro transcription because pCR4TOPO vectors contain a T7 promoter sequence 70bp downstream of the PCR insert, which is flanked by a T7 promoter sequence on both ends. This may result in a mixture of IVT product. Therefore, the esiRNA regions had to be isolated from the plasmids. This
was achieved by digestion using EcoRI restriction enzyme, which cleaves the plasmid backbones 5-‐base pairs upstream and downstream of the esiRNA regions. Following restriction digest, the reaction mixtures were separated by DNA agarose gel electrophoresis (Figure 5.7) and the desired esiRNA regions were purified using the Gel Extraction Kit from QIAGEN.
Concentrations of the purified esiRNA regions were measured using a wavelength NanoDrop 2000. During each round of such preparation, the DNA of esiRNA regions purified are enough for at least 10 in vitro transcription reactions and can be kept at -‐20˚C for long term storage.
Figure 5.8. EcoRI digestion of pCR4TOPO plasmids containing esiRNA region. 3µg plasmids were incubated with EcoRI at 37˚C for 2 hours and samples were resolved on DNA agarose gel. Marked with asterisks are the esiRNA regions.
5.2.3.2 In vitro Transcription
To generate double stranded RNA (dsRNA) from the purified DNA of esiRNA region by in vitro transcription (IVT), the T7 MEGAScript® Kit from Ambion was used. Reactions were set up as was described by Kittler et al. in 2007 (see Chapter 2 for details). The IVT reaction involves binding of T7 RNA polymerase to the T7 promoter
sequence on the 5’ end and subsequent transcription of the esiRNA region. The two resulting single-‐stranded RNAs, which are complementary to each other, are allowed to anneal to each other to give dsRNA. Then, 0.4µl of IVT product was analysed by DNA Agarose gel to check efficiency of the transcription reaction and the rest was used for RNAse III digestion.
Figure 5.9. In vitro transcription of esiRNA region. IVT reaction was set up with 4µl of PCR product and 6µl of reaction mixture from MEGAScript® T7 Transcription Kit (Ambion). 0.3µl of reaction product was analysed on DNA agarose gel.
5.2.3.3 RNAse III Digestion
For enzymatic digestion of the long dsRNA resulting from IVT into short fragments of esiRNAs, the enzyme Ribonuclease III (RNAse III) from Escherichia coli was used. We have received a kind donation from Dr. Dun Yang (UCSF) for the plasmid of RNAse III with a glutathione S transferase (GST) epitope tag (pGEX2T-‐ RNAse III). The sequence of the plasmid was verified and the plasmid was transformed into BL21 competent cells for protein expression. To purify GST-‐RNAse III, I have set up 1L overnight culture of BL21 cells and GST-‐RNAse III expression was induced by isopropyl-‐β-‐D-‐thio-‐galactoside (IPTG; 0,4mM) for 3 hours. After that, bacteria cells were pelleted by centrifugation and lysed by sonification. The cell lysate was then spun at 55000 rpm and GST-‐RNAse III, which is a soluble protein, was pulled down from the supernatant using glutathione-‐agarose beads. Next, GST-‐
RNAse III was retrieved sequentially from the beads and the eluates were combined and dialysed for further purification. Samples were taken at every step during this process and analysed by SDS-‐PAGE to check expression of GST-‐RNAse III, purity of the pulled down product and the extent of degradation. As shown on figure, GST-‐ RNAse III was also found in the cells pellet while remaining largely soluble, and there was no significant degradation of the protein detected. Finally, the protein concentration of the purified GST-‐RNAse III was determined, diluted to 1.5µg/µl and stored at -‐80˚C in 5µl aliquots.
Figure 5.10. Purification of GST-‐RNAse III. (A) GST-‐RNAse III was induced in BL21 cells and was purified from supernatant of the subsequent bacterial lysates, by incubation with glutathione agarose beads at 4˚C for 3 hours. Bound GST-‐RNAse III was eluded sequentially with 1ml elution buffer. Samples were taken for bacterial cells before and after induction, crude lysates after sonification, pellet after centrifugation of lysate and the eluates, and were analysed by SDS-‐PAGE.(Note: Arrow = GST-‐RNAse III). (B). Eluates were combined and dialysed against 4L dialysis buffer. Dialysed samples were analysed by SDS-‐PAGE with different loading volume alongside increasing amount of albumin to determine amount of GST-‐RNAse III.
The dsRNA generated by IVT was incubated with purified GST-‐RNAse III in an esiRNA digestion buffer, with a total volume of 100µl. The digestion mixture was kept shaking at 900 rpm, and for 4 hours at 25˚C followed by 2 hours at 37˚C. 3µl of
A B
the digested samples was analysed by DNA agarose gel electrophoresis to ensure complete digestion, with a single band running around 25bp.
Figure 5.11. RNAse III digested dsRNA. IVT products were incubated with 6µg of RNAse III and in esiRNA digestion buffer. Samples were kept shaking at 900rpm for 4 hours at 25˚C followed by 2 hours at 37˚C. 3µl of digested samples were resolved on 2% DNA agarose gel at 100V for 1 hour.
5.2.3.4 esiRNA Purification
The digested esiRNAs were purified by ion exchange column and precipitated with isopropanol. The esiRNA was spun down by centrifugation and dried in a SpeedVac. The resulting pellet was resuspended in 100µl of RNAse-‐free water and concentration was measured using NanoDrop. 50µl of each of the esiRNAs were distributed on 2 96-‐well plates (Table 5.4), and these serve as a resource for future esiRNA DUB library screens.
Table 5.3
1 2 3 4 5 6 7 8 9 10 11 12
A CYLD DUB3 DUB4 USP1 USP2A USP3 USP4 USP5 USP6 USP7 USP8 USP9X
B -‐ USP10 USP11 USP12 USP13 USP14 USP15 USP16 USP17 USP18 USP19 USP20
C USP21 USP22 USP24 USP25 USP26 USP27X USP28 USP29 USP30 USP31 USP32 USP33
D USP34 USP35 USP36 USP37 USP38 USP39 USP40 -‐ USP42 USP43 USP44 USP45
E USP46 USP47 USP48 USP49 USP50 USP51 USP52 USP53 USP54 USPL1 BAP1 UCHL1
F UCHL3 UCHL5 ATXN3 ATXN3L JOSD1 JOSD2 BRCC3 CSN5 CSN6 EIF3H EIF3S5 MPND
G MYSM1 PRPF8 PSMD
14 PSMD7
STAM BP
STAM