Introduction silence GFP expression, has visualised the action


Introduction

RNA-mediated interference (RNAi) has been widely used to understand gene regulations in C. elegans. RNAi is induced by feeding C. elegans bacteria expressing double stranded RNA (Timmons et al 2001) that complementary to the target mRNA region. Once digested by C. elegans, the dsRNA will be processed by Dicer to produce short interfering RNA(siRNA). The siRNA will associate with RNA silencing complex called RISC which then complementarily binds to the target region of the mRNA. The binding leads to degradation of the mRNA, hence the gene is silenced.

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C. elegans upregulates a class of chitinase-like (chil) gene in their hypodermis. Silencing or knockdown of chil will increase susceptibility of C. elegans to oomycetes. The exact function of Chil genes is yet to be known but it has been hypothesised to act as a first defence barrier for C. elegans. Response to pathogen by C. elegans is monitored by establishing a chil-27 transcriptional reporter, known as chil-27::GFP. Chil-27::GFP will be expressed in the presence of pathogens. This experiment aims to study chromatin factors in C. elegans that involve in the regulation of chil-27::GFP expression by the mean of RNA interference.

 

 

Methods

C. elegans were grown on nematode growth medium(NGM) plates containing oomycete-filtered extract which induces upregulation of chil-27::GFP. The plates were seeded with E. coli HT115 for RNAi treatment.

 

C. elegans used in this experiment is homozygous for the chil-27::GFP reporter. The worms also have rrf-3(pk1426) mutation that causes the dysfunctional of putative RNA-directed RNA polymerase which makes the C. elegans to be hypersensitive to RNA interference (Simmer et al 2002).

 

11 NGM plates were prepared, each seeded with bacterial clones to induce various RNAi that target; 8 chromatin factors, pop-1 which when silenced causes embryonic lethality, unc-22 that leads to uncoordinated movement when disrupted by RNAi, gfp for negative control, pals-22 which knockdown of the gene is known to upregulate chil-27::GFP and empty vector to allow normal chil-27::GFP upregulation which will be used as positive control. 

Three variables measured for data analysis include the level of chil-27::GFP expression, col-12::mCherry expression and observation on morphology of the worms.

 

Result and Discussions  

The negative control works perfectly and allow an accurate benchmark for comparison to the other samples. The observation on the negative control, which uses RNAi that targets to silence GFP expression, has visualised the action of gene silencing by RNA interference. Besides being the negative control for the experiment, it illustrated the ability of RNAi to regulate gene expression, which in this case, it completely silenced the gene responsible for GFP expression. This observation allows visual direct understanding of the role of RNAi in gene silencing.

 

However, the positive control did not meet the expected observation. Instead of near 100% GFP expression, there was 12% GFP expression. The poor chil-27::GFP expression may be caused by contamination or use of the same pipette tips which caused bacterial cross-contamination, leading to disrupted normal chil-27::GFP gene.  Regardless of the presence of a working positive control, the evaluation of other samples treated with RNAi was still carried out using the negative control and by comparing the worm (on the positive control plate) that managed to express chil-27::GFP.

 

C. elegans with RNAi targeted at T24G10.2

GFP expression has been observed in all C. elegans randomly selected from the plates.

Strong induction of chil-27::GFP expression in all C. elegans on this plate suggests silenced of T24G10.2 upregulate the expression of chil-27::GFP.

Gene T24G10.2 is a protein coding gene that involved in reproduction which has been predicted to have a DNA binding activity and acts as transcription co-activator activity (Spencer WC et al. 2011). ??Although the gene predicted function has no direct role in,  the function of T24G10.2 could be argued to be linked to the downregulation of chil-27::GFP gene as the silenced gene caused the host to be have a high level of GFP expression.

 

C. elegans with RNAi targeted at T13F2.2

Another gene silencing that gives to complete chil-27::GFP expression is T13F2.2. T13F2.2 is C. elegans gene responsible for putative RNA polymerase II transcriptional coactivator. Silencing of the gene allows its importance in upregulating chil to be confirmed. It is clear that silencing of this particular gene causes upregulation of chil-27::GFP in C. elegans.

 

K01G5.2 or hpl-2 codes for C. elegans heterochromatin protein 1 (HP1).

Although the silencing of K01G5.2/hpl-2 and Y55B1B_119.b do not lead to a complete GFP expression in every C. elegans screened, they still allowed a significant GFP expression, indicating the genes have a direct link to the upregulation of chil-27::GFP.

 

Only 9 out of 16 worms were observed with GFP expression. Regardless, when compared to the positive control, it still suggests a significant upregulation of chil-27::GFP expression but the level of upregulation could be argued considering the role of K01G5.2/hpl-2. The GFP expression in half of the worms could be due to the interrupted chil-27::GFP expression by hpl-2. The expression of GFP could be due to high efficiency of RNAi in silencing hpl-2, allowing some of the worms to express chil-27::GFP.  Hpl-2 activity is required for various biological functions which include silencing the transgenes in the germ-line, negatively regulates RNAi and it works with other genes (ie hpl-1, lin-13 etc) in C. elegans development.

 

In this particular gene, the gene could disrupt the level expression of chil-27::GFP if RNAi did not effectively silence the gene, causing the accuracy of the level of chil-27::GFP expression to be questioned. However, it is evident that K01G5.2 plays an important role in the upregulation of chil-27::GFP. In general, silencing all 8 chromatin factors caused induction of chil-27::GFP expression when compared to the control. The strength and variablity of the GFP expression suggest that RNAi activity at different regions of C. elegans mRNA can cause different silencing 

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