Genetic Manipulation Using RNA Interferences
Genetic manipulations in songbird brains using virus mediated RNA interference (RNAi) –a short overview prepared by Ezequiel Mendoza, Iris Adam, Jana Petri and Constance Scharff, FU Berlin
Prerequisites:requires a S2 biosecurity lab and adequate permission from the authorities.
Equipment: Laminar flow hood, cell culture incubators, ultracentrifuge, bacterial culture, western blot equipment, FACS, liquid nitrogen, stereotax, microsurgery-injection equipment, Isoflurane vaporisator, high quantities of plasmid vectors.
Rationale: reduce a desired gene product in a spatially and temporally defined manner in organisms where germline transmission is not as efficient as in mice.
How:use stereotaxic surgery to inject a virus that can infect neurons, leading to transcription of sequences resulting in so-called short-hairpin structures (see below). Those are complementary to the target transcripts and will trigger the cellular machinery that results ultimately in the destruction of the transcripts of ‘your’ gene (Dykxhoorn et al., 2003).
Viruses commonly used: Moloney murine leukemia virus (MoMLV) (Koo et al. 2006; Dykxhoorn et al., 2002; Kamihira et al., 2005), murine stem cell virus (MSCV) (Dykxhoorn et al., 2002), and the Lentiviral vectors that are derived from HIV-1 (McGrew et al., 2004; Haesler et al., 2007; Agate et al., 2009; Wada et al., 2006). These types of systems do not activate an interferon response (Dykxhoorn et al., 2002; Elbashir et al., 2001). Promoters used are often U6 and H1 promoters (RNA polymerase III) but polymerase 1 or 2 promoters can also be used.
Hairpin design: The general composition of the short hairpin sequence is: sense sequence èhairpin loop èantisense sequence (an example shown below). Because there are two complementary sequences they hybridize and form a loop. This is recognized by the cellular micro RNA (miRNA) machinery, which incorporates the antisense sequence of the short hairpin and uses it to target the complementary mRNA, which leads to its degradation (Dykxhoorn et al., 2003, Rana et al., 2007).
How do I start designing a short hairpin? The sequence of the desired gene is inserted in different internet tools available for designing short hairpins. We use siRNA design tools of MWG (www.eurofinsdna.com) and Ambion (www.ambion.com). Which one gives better results? The MWG gives results that show the secondary mRNA structure where the short hairpin is going to bind, permitting to see if the region is accessible or not. It also gives a score for the sort hairpin. The Ambion siRNA tool does not show these two features. How good are the short hairpins resulting of both tools? We have short hairpins of both that worked. We usually try both tools and chances are that the hairpins that both agree tend to work. In the past, we have designed 12 short hairpins per gene and after testing them found 2 or 3 that led to dramatically reduced levels of the protein of interest. To control for off-target effects, 3 hairpins per targeted genes are desirable, 2 are a must. Control hairpins usually entail scrambled sequences that do not target a gene in the same species, or e.g. archibacterial sequences. Such a control does not compete with the miRNA machinery since the short hairpin is not targeting. Therefore knocking down a virally overexpressed GFP with a hairpin against GFP is a further control that ensures that activating the entire knockdown cellular machinery, does not cause a confounding effect.
Important: the RNA polymerase III recognizes 4 or more Ts as a termination signal that terminates transcription in the absence of other cofactors. Therefore it is absolutely crucial NOT to include more than 4 consecutive thymidines (uracils) in the short hairpin sequence, which is recognized as a stop signal by the polymerase.
Before you order the oligos of the short hairpins you should check the GC percentage (should be around 50%). You should blast the sequence against the genome to see that they are specific for the desired gene.
Then you design the short hairpins you want to test. We used as the loop sequence the following GTGAAGCCACAGATG, and put some restriction sites before and after the short hairpin, so that the hybridized oligos could be inserted in the pBudΔU6 vector. In order to see if the short hairpin forms the loop one can use internet tools like: www.bioinfo.rpi.edu/applications/mfold
Testing of hairpins: There are many ways to test the short hairpin plasmids in vitro. We transfect cells (usually HeLa) in culture with an over-expression vector of the target gene and add the short hairpin plasmid (usually 1:4 ration). Other studies use as much as 20-fold more short hairpin plasmid than overexpression of target protein. We use Lipofectamine or CalciumPhosphate as transfection agent. Both work well.
Another way to test short hairpins is using the desired protein tagged with GFP or Luciferase. Using GFP gives a fast result, but there is nothing to normalize with. Using Luciferase one can normalize to Renilla or LacZ and one can get a value which reflects the effect of the short hairpin.
To determine successful knockdown in vivo, one or more of the following methods should be used: Q-PCR or western blots of punched out tissue, or in situ hybridization and immunohistochemistry on tissue sections.
Sub-cloning of the working short hairpins into the viral transfer: We use the vector pFUGW_linker. Recombinant lentivirus is generated in our laboratory as follows: HEK293-T cells are used for transfection of viral constructs and titration of virus. Four cell culture plates (10cm diameter CELL+) each containing 8x106 cells with 12ml HEK293-T medium, are transfected with 40 μg viral transfer vector, 20 μg envelop vector pVsVg and 30 μg packaging vector λ8.9 using 225,2 μl Lipofectamine 2000 (Lois et al., 2002; Zufferey et al., 1999). For transfection cells are kept in antibiotic-free cell culture medium. Approximately 4-6 hours post transfection, the culture medium is changed.
Collection of virus: Lentiviral particles can be collected and concentrated through ultracentrifugation after 36h-48h post-transfection. We resuspend the virus pellet after ultracentrifugation in 20μl or less of Hanks' Balanced Salt Solution and incubate (Invitrogen, Carlsbad, USA) overnight at 4°C. Aliquots of e.g. 2μl are shock frozen in liquid nitrogen and stored at -80°C the next day.
Titration of the virus by infection of HEK293-T cells: We determine the virus titer by infection of 4x105 HEK293-T cells, seeded 12hours prior to titration per well of a coated 6-well plate. For infection, 1μl of undiluted, 1:10, 1:100 or 1:1000 diluted virus solution is added directly to the culture medium containing antibiotics. We quantify infection after 72h by flow cytometry. All our virus constructs also encode the green fluorescent protein (GFP), thus the 530nm channel of the FACS is used to determine the number of infected cells. Usually the percentage of green cells in the 1:10 and 1:100 dilutions are used to calculate the titer. The percentage of GFP positive cells are divided by the total number of cells present in the dish before infection (here 4x105) and multiplied with the dilution factor. Titers of virus solution are usually in the range of 1-5x106TU/μl. For certain applications we aim at 107 TU/ μl. The titer of studies that showed a successful reduction range from 1x105 (Eren-Kocak et al.,2011; Mahairaki et al.,2009; Santamarina et al.,2009) to 1x106 TU/µl (Garza et al.,2008; Liu et al.,2011; Haesler et al., 2007; Wada et al., 2006). Higher concentrations can be achieved e.g. by adding a sucrose gradient centrifugation step.
Surgery for virus injections: For local virus injections into birds, we use as a pre-operation painkiller Meloxidyl (active substance is meloxicam, in a dose 0.5 mg per kg body weight) administered with a pipette through the beak 1 hour before operation. We use Isoflurane evaporation as anesthesia mixed with oxygen at the level of 1.5-3% which is delivered to the beak through a pipe system at 1l/min flow rate. We have had good results with this method, having very few fatalities.
With the virus injections, we have seen good results waiting 1 minute after each injection allowing it to spread locally before we move the pipette to the next location.
After the operation and over the next 3 days we give the birds Meloxidyl again, but this time with food once per day to mitigate potential discomfort caused by the surgery.
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