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Lecture 23 (real)

Lecture 23 (real) - 135A223 Lecture 23 24Artificial DNA 1...

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Artificial DNA Artificial DNA 4 3 1 2 2 1 5 4 3 2 1 Lecture 23 Gene Targeting Goal : Go into the genome of an organism and modify organism Method : (1) Disrupting genes (2) Introducing genes (3) Modifying genes Definitions Transgene : Stable incorporation of a gene Make a stable cell line by stable transfection Refers to organism Knock out : Disruption of a specific gene Disrupt it in such a way that there is no gene function Knock in : Replacing a specific gene Go into the genome, take out a specific gene and put another one it Dominant negative : Incorporation of dominant mutant allele causing the same phenotype as loss-of- function Use variants of mutants genes that have the property of antagonising regular activity Some mutants give phenotype when they are heterozygous If dominant, only one allele gives phenotype One mutation of one allele is blocking the whole gene function RNA interference, knock down: Depletion of mRNA Potential to have few mRNAs still around Used in genetic model system to locally disrupt gene activity Always estimate how much of gene product has been removed 1 | Pa g e
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Gene knock out Knock out: Disruption of specific genes Yeast has a habit of liking homologous recombination: Used as a tool to remove genes Sequence you want to target is present: Design a piece of artificial DNA that contains flanking sequences from what you want to remove Example : Remove coding sequence of a particular gene Do PCR product that contains: Identical flanking sequences No gene in the middle Antibiotic resistance gene Result : Disruption construct 2 | Pa g e
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1. Introduce disruption construct into yeast 2. With high efficiency yeast will recombine Recombination : Crossing-over by exchanging endogenous piece for a new piece 1. In one allele, the gene has been replaced by the selection gene Yeast can go from haploid to diploid: As long as they are diploid nothing happens because one allele is sufficient for function 1. Induce sporulation to end up with 4 spores through meiosis Two of the spores should have the mutated gene Only mutated cells have survived: Because the cell has been growing in the presence of antibiotics All the yeast cells present have the knockout because they are alive If gene is essential: Two out of the four will not grow Separate the four spores and observe them: If four grow, and the essential gene was not modified If only 2 grow, an essential gene was modified 3 | Pa g e
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Systematic disruption of 6000 genes in yeast 4500 not required for viability The whole genome of yeast has been tested for this: Only a quarter of the genes are essential When you remove a gene, the animal might be perfectly happy without it: A. Reason 1: Redundancy : Multiple genes of a family that compensate for each other Safety mechanism : If you have a mutant in a gene, another one will cover for it A.
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