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Function in living cells

What does it do in vivo?

Gene encoding the protein

Essential tool for studying proteins of interest.

Mutant cell line/organism

A model lacking protein function used in studies.

Purified protein

Protein isolated for biochemical studies.

Classic microscopy

Traditional imaging methods, including confocal variants.

Biochemistry

The study of chemical processes within and relating to living organisms.

Molecular biology

The branch of biology that deals with the study of molecular basis of biological activity.

Genetics

The study of genes, genetic variation, and heredity in living organisms.

Alleles

Different forms/variants of a gene.

Mutation

A variant allele differing from the wild type.

Wild type

Standard/reference genotype.

Genotype

Genetic constitution of an individual.

Phenotype

Physical appearance/function due to genotype.

Haploid

Having a single set of chromosomes.

Diploid

Having two sets of chromosomes.

Homozygous

Having identical alleles for a specific gene in diploids.

Heterozygous

Having different alleles for a specific gene in diploids.

Synapsis

Homologous chromosome alignment during meiosis.

Crossing over

DNA exchange between homologous chromosomes.

Dominant mutations

Mutations that appear in the F1 generation.

Recessive mutations

Mutations that appear in the F2 generation, typically at a 3:1 ratio.

Haploid/diploid life cycle

Refers to yeast's ability to exist in both forms.

Sporulation

Process that produces 4 haploid spores in yeast.

cDNA Library

A library made from tissue-specific mRNAs.

PCR (Polymerase Chain Reaction)

Technique used to amplify specific DNA sequences.

Sanger Dideoxy Method

DNA sequencing method using dideoxynucleotides for chain termination.

RNA Interference (RNAi)

Process of gene silencing by dsRNA.

DICER

Enzyme that cleaves dsRNA into siRNAs.

CRISPR-Cas9

Genome editing technology that uses a bacterial defense system for manipulation.

E. coli Expression

A method of protein expression with high yields, involving IPTG induction.

Transgenic Organisms

Organisms that have been genetically modified to express foreign genes.

What is the specific application of promoter fusions in transgenic organisms?

To study and visualize gene expression patterns.

What is the specific application of protein fusions in transgenic organisms?

To determine the subcellular localization of a protein.

What is the purpose of GFP tagging for live imaging?

To visualize proteins in living cells or organisms.

What technique is commonly used for gene disruption in yeast?

PCR-mediated gene replacement.

How are successful gene replacements identified in yeast gene disruption?

Through the use of selectable markers, such as G-418 resistance.

How are essential genes identified in yeast?

Via spore viability analysis after gene disruption.

What are cell type-specific knockouts?

Gene knockouts that are restricted to specific cell types or tissues.

How is conditional gene deletion achieved in the Cre-loxP system?

The Cre recombinase recognizes loxP sites flanking a gene, excising the gene only in cells expressing Cre.

What is dominant-negative expression?

A technique where a mutated protein interferes with the function of the wild-type protein.

Which Nobel Prize was awarded for CRISPR-Cas9 genome editing?

The 2020 Nobel Prize in Chemistry.

What are the main components of the CRISPR-Cas9 system?

Cas9 protein (DNA nuclease), guide RNA (sequence targeting), and template DNA (for repair specification).

List some applications of CRISPR-Cas9 technology.

Gene correction for disease treatment, creation of specific mutations for research, gene deletions for functional studies, and protein tagging for localization.

What is the basis of mouse gene knockouts?

Embryonic Stem (ES) Cell Technology, utilizing totipotent ES cells derived from blastocysts.

How are modified ES cells selected during mouse gene knockout procedures?

Through selection for rare homologous recombination events in culture.

What is chimera production in the context of mouse knockouts?

The process of injecting modified ES cells into host blastocysts to create chimeric mice, which can then transmit the modified gene through the germline.

Who discovered dsRNA-induced gene silencing, leading to the 2006 Nobel Prize?

Andrew Fire and Craig Mello.

What is the role of the RISC complex in RNAi?

It incorporates siRNAs and, along with the Argonaute protein, targets complementary mRNA for degradation, leading to gene silencing.

Beyond gene function analysis, what are other applications of RNA interference?

Large-scale screens targeting every gene, therapeutic potential for disease treatment, and roles in virus defense and transposon silencing.

What are common delivery methods for dsRNA in RNAi experiments?

Injection into tissues, feeding E. coli expressing dsRNA, or transcription of inverted repeats within the organism.