1

Tanyut Huidrom

Professor Anup Padmanabhan

BIO - 2202

24 April 2023

Assignment 3

AIM

The aim of this experiment is to investigate the potential effects of double-stranded RNA

(dsRNA) targeting Par5, a gene responsible for maintaining the orientation of the mitotic spindle

during cell division, and the effects of it’s knock down on the viability of c. elegans embryos.

PRINCIPLE

The basic principle of RNAi in C. elegans involves the use of double-stranded RNA (dsRNA)

molecules that are complementary to the mRNA transcript of a target gene. When these dsRNA

molecules are introduced into C. elegans cells, they are processed by the enzyme Dicer into

small interfering RNAs (siRNAs), which are typically 21-25 nucleotides long. The siRNAs then

bind to an RNA-induced silencing complex (RISC), which uses the siRNA as a guide to identify

and bind to the target mRNA. The RISC then cleaves the mRNA, leading to its degradation and

the silencing of gene expression. RNAi in C. elegans can be transmitted from one generation to

the next, a process known as systemic RNAi. This occurs when dsRNA is introduced into the

germline of C. elegans, either by injection or by feeding the worm bacteria expressing dsRNA.
 2

The siRNAs produced by the germline cells are then transmitted to the developing embryos,

where they can silence gene expression in a highly specific manner.

● RISC: RNA-induced silencing complex (RISC) is a multisubunit protein complex that is

responsible for binding small RNA molecules, such as siRNA and miRNA, and using

them to target specific mRNA molecules for degradation. RISC contains several key

components, including Argonaute proteins, which are responsible for cleaving the

targeted mRNA molecules, and several auxiliary proteins that help to stabilize the

complex and facilitate its interaction with RNA.

● DICER: DICER is an enzyme that cleaves double-stranded RNA (dsRNA) molecules

into small interfering RNAs (siRNAs) and microRNAs (miRNAs). DICER is a member

of the RNase III family of nucleases, and contains two RNase III domains that are

responsible for cleaving the dsRNA molecule into smaller fragments. It also contains

several other domains that are involved in binding and processing RNA molecules, as

well as a helicase domain that helps to unwind the dsRNA.

● Argonaute complex: The Argonaute complex is responsible for cleaving the targeted

mRNA molecules during the RNAi pathway. The Argonaute protein is a member of the

PAZ/PIWI family of proteins and contains two main domains: the PIWI domain, which is

responsible for cleaving the mRNA molecule, and the PAZ domain, which is involved in

binding small RNA molecules, such as siRNA and miRNA. The Argonaute protein

interacts with the small RNA molecule to form the RISC complex, which then scans the

mRNA molecule for complementary sequences, leading to its cleavage and degradation.
 3

● siRNA: siRNA (small interfering RNA) is a type of small RNA molecule. siRNAs are

typically 20-25 nucleotides in length and are produced by the cleavage of

double-stranded RNA (dsRNA) by the enzyme Dicer. Once produced, siRNAs are

incorporated into the RNA-induced silencing complex (RISC), which is responsible for

targeting specific mRNA molecules for degradation or translational repression. The

siRNA binds to complementary mRNA sequences, leading to cleavage of the mRNA

molecule by the endonuclease activity of the Argonaute protein within the RISC

complex.

● miRNA: miRNA stands for microRNA, which is a type of small RNA molecule that

plays a key role in post-transcriptional gene regulation. miRNAs are typically 20-25

nucleotides in length and act by binding to specific mRNA molecules, leading to their

degradation or translational repression. miRNAs are transcribed from genomic DNA and

are processed by the enzyme DICER to form mature miRNA molecules. miRNAs have

been implicated in a wide range of biological processes, including development, cell

differentiation, and disease.

● dsRNA: dsRNA stands for double-stranded RNA, which is a type of RNA molecule that

contains two complementary RNA strands that are paired together. dsRNA is processed

by the enzyme DICER to form small interfering RNAs (siRNAs) and microRNAs

(miRNAs). dsRNA can also be used as a tool for gene silencing, as it can be introduced

into cells to trigger the RNAi pathway and silence specific genes.

● IPTG: IPTG (isopropyl β-D-1-thiogalactopyranoside) is a chemical inducer that can be

used to induce the expression of genes of interest in bacterial cells, including genes
 4

encoding double-stranded RNA (dsRNA). In plasmids, IPTG is commonly used to induce

the expression of a T7 RNA polymerase gene, which in turn can produce dsRNA from a

gene of interest under the control of a T7 promoter.

MATERIALS

● Fridge

● Plasmid Containing Par5 gene (HT1115 (DE3))

● L4440 Plasmid

● AT Plates

● Luria Broth

● Ampicillin

● Tetracycline

● Test Tubes

● IPTG

● NGM Plates

● OP50

● Washing Solution

● Worm Pick
 5

METHOD

Plasmid Preparation:

1. The bacteria in question are stored in a fridge at -80oC. We are using HT1115(DE3) to

knock down the Par5 gene, and an empty vector (L4440) as our control.

2. Take 2 AT plates containing LB, Ampicillin and Tetracycline. Streak the bacteria onto the

plates and keep them overnight/till colonies appear.

3. Take one colony from each plate and add them into a test tube with LB in order to create

a primary. Add Ampicillin to your primaries and incubate for 12 hours at 37oC.

4. Take a sample from both primaries and move both to new test tubes containing LB and

Ampicillin. Add IPTG after 3 hours. These are your secondaries.

5. After 3 hours, take samples from the secondaries and add them to NGM plates containing

IPTG and Ampicillin.

Worm Preparation:

1. We bleached and synchronized 20-30 worms and let them reach L4.

2. We then washed the worms to remove the OP50 growth medium.

3. The worms were then moved to the prepared plasmid plates (12-14 worms per plate)

using a worm pick.

4. After 48 hours, they were moved to blank plates and we let them lay eggs.

5. We counted the eggs and sacrificed the mothers to prevent more eggs from being laid.

This is our T0 count.

6. We counted the eggs again the following day. This was our T1 count.
 6

RESULTS

Embryo Counts T0 T1 Embryonic Lethality

L4440 266 23 8.64%

Par5 235 168* 71.48%

INFERENCE

We were expecting an embryonic mortality rate of 95-100% for the Par5 knock down group of

worms but the observed mortality rate was ~70%. While there could have been other factors at

play in the reduced mortality of the embryos, such as incomplete knock down of the gene or

compensatory mechanisms in other genes, the most probable culprit behind this discrepancy was

variability in the experimental conditions. When the embryonic counts were taken at T0, it was

discovered that 5 mothers had embedded themselves in the media because of lesions made in the

media surface during handling, and it was not possible to sacrifice them all at T0. Our TA’s told

us that they would try to remove them using other methods but ultimately 1 mother was left alive

in the Par5 plate due to being hidden along the sides of the plate and media. However, despite

this error in the experimental setup, we are able to see a clear distinction in the degree of

embryonic lethality showcased by the Par5 setup vs. our control of L4440. This would suggest

that Par5 plays an essential role in embryonic development, likely related to its involvement in

asymmetric cell division, spindle orientation, and cytokinesis. The observed increase in

embryonic lethality could indicate that knockdown of Par5 has disrupted the proper orientation

of the mitotic spindle during cell division, leading to abnormalities in embryonic development
 7

and possibly resulting in embryonic death. Alternatively, knockdown of Par5 could have

disrupted the proper distribution of developmental factors between the two daughter cells,

leading to abnormal development and ultimately embryonic lethality. Overall, the increased rate

of embryonic lethality observed in c. elegans with Par5 gene knocked down suggests that Par5 is

an essential gene for embryonic development in c. elegans and that its function is critical for

proper cell division and developmental processes.