This method was used in order to help determine which transcriptional factors cause the F45D3.4 gene to be expressed. There were 387 transcription factors in the round worm that needed to be looked at. To determine which of these were actually causing the gene to turn "on", each transcription factor needed to be inactive. Once inactive, the affect of the factor on the gene expression could be observed. If the factor affects the gene expression, the gene expression will be inhibited because the factor is needed for it to be active.

In order to knock down or inactivate each of the transcription factors, a procedure known as RNA interference (RNAi) is used. This procedure uses double-stranded RNA (dsRNA) which acts as a signal to silence the expression of specific genes in organisms when it is introduced. dsRNA acts as a signal through a pots-transcriptional mechanism, meaning it destroys the RNA that transcribes the targeted gene [1].

So how is dsRNA created? The first step is to identify the transcription factor of interest and its DNA sequence. Once this sequence is matched to its appropriate primer (a short DNA segment that pairs complementary to the sequence of interest), the primer can bind and DNA-polymerase will synthesize the double stranded DNA and amplify the amount of this transcription factor (a detailed explanation of how primers work can be viewed in the support page of qPCR) [2]. This PCR product can then be placed into a L4440 RNAi feeding vector. This feeding vector is what is ultimately going to help knock down the transcription factors.

Figure 1: Incorporation of the cloned product (transcription factor) into the L4440 RNAi feeding vector [4].

How is the PCR product put into the vector, and what exactly is a vector? A vector is a DNA molecule that can be used to carry and then transfer foreign genetic material into another cell. Once it is transferred into the cell, it can be replicated and expressed [3]. The vector is cut by a restriction enzyme, called EcoRV (represented by the scissors in figure 1). It is cut in such a way that there are two thymine nucleotides that hang off in each end. These thymine nucleotides are complementary pairs to the two adenosine nucleotides that are present on the PCR product. This allows for the PCR product to insert itself into the vector. DNA ligase is then able to seal the strands together and create the new vector that contains the transcription factor.

Figure 2: The L4440 RNAi feeding vector with the PCR product and T7 promoters labeled [4].

Once the PCR product is loaded into the feeding vector, the vector can be added to the bacteria strain (this is where the foreign genetic material will be replicated and expressed). The question of interest now, is how is the PCR product transcribed and how do we ultimately end up with dsRNA so that the transcription factor of interest can be knocked down? On each end of the PCR product is something called a T7 promoter, which can be seen in figure 2. This promoters is important as it is the location where the bacteriophage T7 RNA polymerase is able to bind.

What is bacteriophage T7 RNA polymerase? This specific type of RNA polymerase will bind to the promoters identified in figure 2, and will synthesize mRNA for the PCR product. It is a viral polymerase and therefore it has its own promoter sequence and when introduced into the bacteria, it will take over and be the only type of RNA polymerase. This allows for the bacteria to only be used for the transcription and translation of the dsRNA of the PCR product. So how does T7 RNA polymerase become active? The bacteria strain that the feeding vector is added to is E. coli strain HT115(DE3). This specific strain contains a modified lac promoter that controls the transcription of T7 RNA polymerase.

Figure 3: The synthesis of T7 RNA Polymerase.

This means that it contains a sequence of DNA that when activated, will be transcribed and then translated to produce the protein T7 RNA polymerase. Under normal conditions, the sequence that produces T7 RNA polymerase is not transcribed as it is blocked by a repressor. In order to become active, a compound called allolactose or IPTG is added. When allolactose or IPTG binds to the promoter region for T7 RNA polymerase (as seen in figure 3), the T7 RNA polymerase protein is eventually synthesized [5].

Figure 4: An illustration of dsRNA being synthesized [6].

T7 RNA polymerase is highly selective for its own promoter sequences, and the only promoter sequence present in the E.Coli is in the feeding vector. Therefore, it will synthesize RNA for the PCR product and it does so at an extremely rapid pace. Due to there being promoters at each end of the PCR product, the RNA will be synthesized as double stranded RNA (dsRNA). This can be seen in figure 4. Each bacterium that is created now has specific dsRNA's which will target their given transcription factors and knock them down. But how do the bacteria get into the round worms and do this?

The bacterium containing the dsRNA are fed to the round worms by being put into an agar which is dispensed into the same plate that the worms grow in. From the feeding process, the worms are introduced to the dsRNA which will then knock down the specific transcription factor in the worm.

Figure 5: An illustration of dsRNA knocking down the gene of interest.

How does dsRNA actually knock down the genes we are targeting? This is what the RNA interference method does. Once we have the dsRNA, it is able to be cut into small pieces. These small pieces are called small interfering RNAs (siRNA). siRNA's bind to proteins called argonaute proteins, which removes one of the strands of the dsRNA. This free strand is able to bind to mRNA of the specific transcription factor that is being targeted as it is a complementary pair. Once bound, the argonaute protein can cleave the mRNA, destroying it. This causes the transcription factors of interest to never be synthesized and therefore remain inactive [7]. An illustration of this can be seen in figure 5.

1. Sharp, P. A. (1999, January 01). RNAi and double-strand RNA. Retrieved from http://genesdev.cshlp.org/content/13/2/139.full.html

2. Huggett, J., & O'Grady, J. (2014, May). PCR Primers. Retrieved from https://www.highveld.com/pcr/pcr-primers.html

3. Lodish, H. (1970, January 01). DNA Cloning with Plasmid Vectors. Retrieved from https://www.ncbi.nlm.nih.gov/books/NBK21498/

4. Kamath, R. (2003). Genome-wide RNAi screening in Caenorhabditis elegans. Methods,30(4), 313-321. doi:10.1016/s1046-2023(03)00050-1

5. Tabor, S. (2001). Expression Using the T7 RNA Polymerase/Promoter System. Current Protocols in Molecular Biology. doi:10.1002/0471142727.mb1602s11

6. DNA Interactive: Discovering the DNA Structure and beyond. (n.d.). Retrieved from http://www.dnai.org/

7. How RNAi Works - RNAi Biology | UMass Medical School. (2013, November 03). Retrieved from https://www.umassmed.edu/rti/biology/how-rnai-works/