Cell Culture Techniques Lecture 7+8

RNA interference (RNAi) is a natural process in cells where small RNA molecules (siRNA) target and degrade specific messenger RNA (mRNA) molecules, leading to a reduction in the production of the corresponding protein. This process is essential for regulating gene expression and defense against viruses.

In RNAi, small interfering RNA (siRNA) molecules are designed to be complementary to a specific target mRNA sequence. These siRNAs are introduced into cells, where they are incorporated into the RNA-induced silencing complex (RISC). The siRNA guides RISC to the target mRNA, leading to its degradation and reduced protein production.

RNAi can be used to study the function of genes by reducing the expression of a gene-of-interest. This allows researchers to observe the effects of reducing the protein product encoded by that specific gene. This approach can be used in research and potentially for therapeutic purposes.

While RNAi is generally effective, it's important to be aware of potential unintended consequences. These can include off-target effects, where siRNAs might interact with other unintended mRNAs, or alterations in related biological pathways, leading to unexpected changes in cellular function.

When using RNAi, it's crucial to consider various factors that can influence results. These include the cell type, the time point of observation, the specific assay used, and the overall cellular context. Different experimental conditions can lead to variations in outcomes.

RNA interference (RNAi) is a natural process in cells that uses small RNA molecules to silence gene expression. It offers a powerful tool to study gene function by reducing the levels of specific proteins.

A technique used to knock down (reduce) the expression of a target gene. It is based on the principle of RNA interference (RNAi).

Small interfering RNA (siRNA) is a type of double-stranded RNA molecule used in RNA interference (RNAi) to silence specific genes by targeting their messenger RNA (mRNA).

Short hairpin RNA (shRNA) is a type of RNA molecule that folds into a hairpin shape. It is used to silence gene expression by targeting its corresponding mRNA.

This method uses genetic tools to alter the DNA sequence of a cell, potentially causing a permanent change in the cell behavior. It can be used to 'knock out' a gene, meaning the gene is completely deleted, or to introduce specific mutations.

This method utilizes a reporter gene, which produces a detectable signal, to monitor the activity of a specific gene/protein. This allows scientists to understand how the gene/protein functions in the cell.

This method involves introducing an increased copy number of a gene into a cell. This leads to overproduction of the targeted protein, allowing scientists to study its effects and function.

Apoptosis is a programmed cell death process, which is a natural and regulated way for cells to die without causing harm to the surrounding tissues. It is a vital process for development, tissue homeostasis, and defense against disease.

A type of genome editing tool that uses a protein (Cas9 nuclease) to make precise cuts in DNA, allowing for targeted modifications.

A double-stranded break (DSB) in DNA is a serious injury that can be repaired by two main pathways: non-homologous end joining (NHEJ) and homology-directed repair (HDR).

A repair mechanism that joins broken DNA ends together, often with some DNA lost or added, leading to unpredictable changes. This is useful for creating knockout genes.

A more precise repair mechanism that uses a template to copy information and fill in the gaps in broken DNA. This allows for targeted insertions, mutations, or gene replacements.

A type of RNA molecule that guides the Cas9 nuclease to the target DNA sequence for cutting. It's like a GPS for genome editing.

A bacterial protein that can cut DNA precisely at a specific location determined by the guide RNA. This is the 'cutting tool' in CRISPR-Cas9.

The process of using CRISPR-Cas9 to edit genes in living cells. This can be used for research, developing new therapies, and modifying crops.

A type of CRISPR system using a protein called Cas13 that targets RNA instead of DNA. This has potential in antiviral therapy by targeting viral RNA.

A technique used to permanently modify a gene's sequence within a cell, potentially altering the protein it produces. This involves introducing a donor DNA sequence with the desired modification, alongside an enzyme (Cas9) that cuts the target gene at a specific location based on a guide RNA.

A type of KI that alters the protein by inserting or deleting a sequence, leading to changes in its function.

A KI strategy that adds a new sequence at either the beginning (N-terminal) or end (C-terminal) of the gene.

A specialized type of KI that introduces a fluorescent protein (like GFP) to tag a specific protein within a cell.

A method in fluorescent protein KI where the fluorescent protein is linked directly to the target protein.

A method in fluorescent protein KI where the fluorescent protein is expressed from the same mRNA as the target protein, but translated separately.

A sequence within a bicistronic mRNA that causes the ribosome to skip ahead to a second start codon, allowing two separate proteins to be translated.

A self-cleaving peptide sequence used in bicistronic systems to separate two proteins after translation.

Coding regions within a gene that are transcribed and translated into protein.

Non-coding regions within a gene that are transcribed but not translated into protein. They play crucial roles in regulating gene expression and splicing.

Regulatory regions located at the 5' and 3' ends of a gene that influence translation. They control how much and where a protein is made.

A technique used to disable or modify the function of a gene by introducing a random insertion or deletion (indel) into its sequence. This can lead to a premature stop codon, truncating the protein.

This strategy involves using two guide RNAs to delete one or more exons from a gene. This can lead to a truncated or non-functional protein depending on the exons deleted.

A strategy for creating a gene knock-out that uses two guide RNAs to delete an exon and a nearby intron. This results in an exon-intron fusion, disrupting protein production and often leading to protein degradation.

A process involving multiple steps, from designing guide RNAs to analyzing edited cell populations, to permanently modify a gene in a cell line for research or therapeutic applications.

This timeline outlines the steps and estimated time required for conducting a gene knock-out experiment using CRISPR technology, from designing guide RNAs to selecting successful clones.

dCas9 is a modified version of the Cas9 protein that lacks the ability to cut DNA. Instead, it can be fused with other proteins, allowing it to manipulate gene expression or perform other functions without altering the DNA sequence.

CRISPRa refers to CRISPR-mediated activation of gene expression. dCas9 is fused with activator proteins, leading to increased transcription of target genes. This can be achieved through direct interactions with the transcriptional machinery or indirectly by modifying the chromatin landscape.

CRISPRi refers to CRISPR-mediated inhibition or repression of gene expression. In this approach, dCas9 is fused with repressor proteins, leading to decreased transcription of target genes. It can work by directly blocking transcription or by influencing the chromatin structure.

Epigenome editing is the modification of epigenetic marks, such as DNA methylation or histone modifications, using dCas9 fused with enzymes that modify these marks. This allows for targeted changes in gene expression without altering the DNA sequence.

DNA labeling involves using dCas9 fused with a bioluminescent protein like NanoLuc to visualize specific DNA sequences within cells. This allows for real-time monitoring of gene expression and localization.

A pooled screen is a type of genetic screening experiment where a large library of CRISPR/Cas9 constructs, targeting different genes, is introduced into cells. The cells are then exposed to a particular condition (e.g., a drug treatment) and the surviving cells are analyzed to identify genes that contribute to the observed phenotype.

An arrayed screen is another type of genetic screening experiment, where the CRISPR/Cas9 constructs are individually delivered into cells in a controlled array (usually in plates with multiple wells). This allows for a more precise study and analysis of the effects of individual gene modifications.

CRISPR/Cas9 technology can be used to study various cellular responses, such as cell proliferation, differentiation, senescence, cell death (apoptosis or necrosis), and metabolic changes. This allows researchers to understand the roles of different genes in these processes and how they are affected by external stimuli or disease conditions.

The 'cycle' of cellular processes refers to the different stages a cell goes through in its life, including growth, differentiation, division, and eventually, death. Studying these processes using CRISPR/Cas9 helps us understand the dynamics of cellular life.

CRISPR/Cas9 technology can be used to investigate disease mechanisms, identify drug targets, and develop personalized therapies. It allows researchers to modify the genome, study gene function, and understand the interplay between genes and diseases. This knowledge can be used to develop more effective treatments.