knockouts lecture

8/2/2019 Knockouts Lecture

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Outline

Detection of Nucleic Acids andProteins

Gene Function in Eukaryotes


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Recombinant DNA

Bacteriophage vectors can accommodatelarger fragments of insert DNA.

Sequences that arent needed for virus

replication are removed and replaced withunique restriction sites for insertion ofcloned DNA.

The recombinant molecules are then putinto E. coli, where they replicate to yieldmillions of progeny phages containing asingle DNA insert.


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Recombinant DNA

For even larger fragments of DNA, 5major types of vectors are used.

1. Cosmid vectors containbacteriophage sequences, origins ofreplication, and genes for antibioticresistance, so they are able to replicate

as plasmids in bacterial cells.


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Recombinant DNA

2. Bacteriophage P1 vectors allowrecombinant molecules to be packagedin vitrointo P1 phage particles and be

replicated as plasmids in E. coli.

3. P1 artificial chromosome (PAC)vectors also contain sequences of

bacteriophage P1 but are introduceddirectly as plasmids into E. coli.


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Recombinant DNA

4. Bacterial artificial chromosome(BAC)vectors are derived from anaturally occurring plasmid of E. coli

(the F factor).5. Yeast artificial chromosome (YAC)vectors contain yeast origins of

replication and other sequences thatallow them to replicate as linearchromosome-like molecules in yeastcells.


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Detection of Nucleic Acids and Proteins

Recombinant DNA libraries arecollections of clones that contain all thegenomic or mRNA sequences of a

particular cell type.

A genomic library of human DNA can bemade by cloning random DNA

fragments of about 15 kb in a vector.


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Figure 4.26 Screening a recombinant library by hybridization (Part 1)


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Figure 4.26 Screening a recombinant library by hybridization (Part 2)


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In addition to traditional genetic screensfor new mutations, cloned DNA can beused to create transgenic or geneknock-out systems

Yeasts are used in studies of eukaryoticcells because they are easily grown inculture, reproduce rapidly, and have asmall genome.

Mutants that have specific nutrient

requirements can be easily isolated.


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A gene corresponding to any yeastmutation can be cloned, simply on thebasis of its functional activity.

Yeast genes encoding a wide variety ofessential proteins have been identifiedin this manner.

In many cases, such genes have alsobeen useful in identifying and cloningrelated genes from mammalian cells.


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Figure 4.33 Introduction of DNA into animal cells


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Direct microinjection into the nucleus

Coprecipitation of DNA with calciumphosphate to form small particles that are

taken up by the cells Incorporation of DNA into liposomes that

fuse with the plasma membrane

Exposure of cells to a brief electric pulsethat opens pores in the plasma membrane(electroporation)

Viruses


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Figure 4.34 Retroviral vectors (Part 1)

Fi R i l (P )


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Figure 4.34 Retroviral vectors (Part 2)


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Cloned genes can also be introducedinto the germ line of multicellularorganisms.

Mice that carry foreign genes(transgenic mice) are produced bymicroinjection of cloned DNA into the

pronucleus of a fertilized egg.

Fi 4 35 P d ti f t i i


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Figure 4.35 Production of transgenic mice


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Embryonic stem (ES) cells are anotherway to introduce cloned genes intomice.

Cloned DNA is introduced into ES cellsin culture, then stably transformed cellsare introduced back into mouse

embryos.

Fi 4 36 I t d ti f i t i i b i t ll (P t 1)


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Figure 4.36 Introduction of genes into mice via embryonic stem cells (Part 1)

Figure 4 36 Introduction of genes into mice via embryonic stem cells (Part 2)


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Figure 4.36 Introduction of genes into mice via embryonic stem cells (Part 2)


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The ability to introduce specificmutations into cloned DNAs (in vitromutagenesis) is a powerful tool instudying the expression and function ofeukaryotic genes.

Sometimes called reverse genetics,

since a mutation is introduced into agene first and its functionalconsequence is determined second.


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The most common method of in vitromutagenesis uses syntheticoligonucleotides to generate changes

in a DNA sequence.

In vitromutagenesis allows detailedcharacterization of the functional roles

of both regulatory and protein-codingsequences of cloned genes.

Figure 4 38 Mutagenesis with synthetic oligonucleotides


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Figure 4.38 Mutagenesis with synthetic oligonucleotides


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To determine the role of a cloned gene,the activity of the normal gene copymust be eliminated.

Gene inactivation by homologousrecombination: the mutated copy ofthe cloned gene replaces the normalgene copy in the chromosomal DNA.

This occurs frequently in yeast but israre in mammalian cells.


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Yeasts are used in studies of eukaryoticcells because they are easily grown inculture, reproduce rapidly, and have asmall genome.

Mutants that have specific nutrientrequirements can be easily isolated.


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Temperature-sensitive mutantsencode proteins that are functional atone temperature (permissivetemperature) but not another(nonpermissive temperature).

The ability to isolate temperature-sensitive mutants has allowedidentification of genes controlling manyfundamental cell processes.

Figure 4 32 Screening for Temperature sensitive mutations in yeast


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Figure 4.32 Screening for Temperature sensitive mutations in yeast


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Genes can be readily inactivated by

homologous recombination in mouseembryonic stem cells

Stem cells can be introduced into

embryo to make chimeric mice

These mice can be bred to yield progenywith mutated copies of the gene onboth homologous chromosomes.

The effects of inactivation of a gene canthen be investigated in the context ofthe intact animal.

Figure 4 40 Production of mutant mice by homologous recombination in ES cells


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Figure 4.40 Production of mutant mice by homologous recombination in ES cells

Ensuring homologous recombination


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Ensuring homologous recombination

NeoR

HSVtk

NeoR

Homologous

recombination

NeoR+/ HSVtk-

Random integration

NeoR+/ HSVtk+

HSVtk will convert

gancyclovir into a toxic

drug and kill HSVtk+ cells


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Homologous recombination has beenused to systematically inactivate(knockout) every gene in yeast.

A collection of genome-wide yeastmutants is available for scientists touse to study the function of any desired

gene.


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Other approaches are used to interferewith gene expression or function.

Antisense nucleic acids are RNA orsingle-stranded DNA complementary tothe mRNA of the gene of interest(antisense).

They hybridize with the mRNA and blockits translation into protein.

Figure 4.41 Inhibition of gene expression by antisense RNA or DNA


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g g p y


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RNA interference (RNAi)was firstdiscovered in C. elegansin 1998, whenFire and Mello found that injection ofdouble-stranded RNA inhibitedexpression of a gene with acomplementary mRNA sequence.

Double-stranded RNA resulted in extensivedegradation of the target mRNA, whereassingle-stranded antisense RNA had onlya minimal effect.

Key Experiment 4.2 RNA Interference:


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y p


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Southern blotting is widely used fordetection of specific genes.

DNA is digested with a restriction

endonuclease, and the fragmentsseparated by gel electrophoresis.

The gel is then overlaid with a nitro-

cellulose or nylon membrane to whichthe DNA fragments are transferred(blotted). The filter is then incubated witha labeled probe.

Figure 4.25 Southern blotting


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g g


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Northern blotting, a variation ofSouthern blotting, is used for detectionof RNA instead of DNA.

It is often used in studies of geneexpression, for example, to determinewhether specific mRNAs are present.


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Any gene for which a probe is availablecan then be isolated from such arecombinant library.

cDNA clones can be used as probes toisolate the corresponding genomicclones, or a gene cloned from onespecies (e.g., mouse) can be used toisolate a related gene from a differentspecies (e.g., human).


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Hybridization to DNA microarraysallows tens of thousands of genes to beanalyzed simultaneously.

A DNA microarray is a glass slide ormembrane filter onto whicholigonucleotides or fragments of cDNAs

are printed by a robotic system in smallspots at a high density.


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One application of DNA microarrays is instudies of gene expression; forexample, a comparison of the genes

expressed by two different types ofcells.

Figure 4.27 DNA microarrays


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In situhybridization can be used todetect homologous DNA or RNAsequences in cell extracts,

chromosomes, or intact cells.

Hybridization of fluorescent probes tospecific cells or subcellular structures is

analyzed by microscopic examination.

Figure 4.28 Fluorescence in situhybridization


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knockouts lecture

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