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Knockout Mouse: A transgenic mouse in which the normal gene is missing or engineered so that is not transcribed or tran PowerPoint Presentation
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Knockout Mouse: A transgenic mouse in which the normal gene is missing or engineered so that is not transcribed or tran - PowerPoint PPT Presentation


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Transgenic Mouse: Generic term for an engineered mouse that has a normal DNA sequence for a gene replaced by an engineered sequence or a sequence from another organism.

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Presentation Transcript
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Transgenic Mouse: Generic term for an engineered mouse that has a normal DNA sequence for a gene replaced by an engineered sequence or a sequence from another organism.

Knockout Mouse: A transgenic mouse in which the normal gene is missing or engineered so that is not transcribed or translated. “Knocks out” that gene.

Knockin Mouse: A transgenic mouse in which the engineered “transgene” is subtly manipulated to: (A) alter the function of the gene (e.g., replace one amino acid with another in a site to determine if that site is essential for the protein’s function); (B) change transcription rate to overproduce or underproduce the gene product; or (C) create a fluorescent gene product to map its distribution in tissue.

Conditional Knockout (Knockin) Mouse: A transgenic mouse in which the transgene is knocked out (or in) in specific tissues, at a specific developmental stage, or in response to an exogenous substance (e.g., an antibiotic).

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Transgenic Organisms

General Outline:

  • Infect blastocyst cells/sperm with viral vector with the gene of interest.
  • Hope that in some cells homologous recombination will insert the DNA section of interest into the target cell’s chromosome.
  • Select chimeric organisms.
  • Breed until the transformed DNA is found in a germ line.
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agctta

cgatc

agctta

cgatc

Gene of Interest

tcgaat

gctag

tcgaat

gctag

Downstream

DNA (unique)to the gene,usually > 1 kb

Upstream

DNA (uniqueto the gene),usually > 1kb

Desired Gene

Antibiotic Resistance Gene

(1) Get the nucleotide sequence of the gene of interest. Including upstreamand downstream nucleotides.

(2) Construct the desired DNA sequence (i.e., the transgene), adding a gene for antibiotic resistance, but keeping the upstream and the downstream nucleotides.

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(3) Micropipette embryonic stem cells from the inner cell mass of ablastocyst (i.e. early mouse embryo) in a strain with a physically recognizablephenotype (e.g., pigmented).

(4) Culture the cells with many copies of the manufactured transgenicDNA complex. Short bursts of an electrical current allow the DNA to passthrough the plasma membrane into the cell (electroporation).

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+ antibiotic

(5) Cells will divide in culture and some of them will incorporate the transgenic DNAstrand into the chromosome (homologous recombination). After a sufficient numberof cell divisions, add the antibiotic. This will preferentially kill those stem cells thathave not incorporated the transgenic strand (black dots), giving a good harvest ofthose that have incorporated the strand (red dots).

(6) Insert the stem cells into the blatocyst of a mouse with a different geneticbackground trait (e.g., an albino if the original stem cells came from a pigmented mouse).

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(7) Implant the new blastocysts into a pseudopregnant female with a visiblephenotype different from the blastocyst phenotype (e.g., albino if the blastocyst is pigmented).

(8) Offspring that have pigmented sections are chimeras that have incorporatedthe transgenic sequence into their cell lines. Select them for further breeding.

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(9) Keep breeding the offspring of the chimeras until some fully pigmented miceare born. A fully pigmented mouse means that the transgenic germline generatedone of the gametes that resulted in that mouse. Genotype the mouse to determinethe genotype at the desired locus and the insertion point(s). (Most will be heterozygotes for the wild type allele and the transgenic allele).

(10) Mate two heterozygotes and genotype their offspring. This will give all threegenotypes--wild type homozygotes, heterozygotes, and transgenic homozygotes.

(11) Compare the three genotypes on the phenotype of interest.

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(12) Problems:

Multiple insertions: too much protein.

Insertion into a life-necessary gene: lethality.

Insertion into a gene leading to gene-silencing: no protein.

Insertion in a different area can lead to differential gene regulation.

Background genotype can be limiting.

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Conditional Gene Expression

Generate a transgenic line. Engineer the transgene section so that it contains unique sections (a loxP site) to a specific Cre recombinase (enzyme that catalyzes recombination) at the sites.

Mate this line with the desired Cre recombinase mouse strain. E.g., one that has the Cre recombinase in the hippocampus. There is a very large number of Cre recombinase mouse lines, each with tissue-specific, developmentally specific, or exogenous chemical specific properties (e.g., responds to tetracycline).

The offspring will then have the gene knocked out in the desired tissue, at the desired developmental stage, or when the chemical inducer is administered. The knockout occurs when cells divide and the transgene “loops” during recombination and is not included in the recombinant chromosome.

NOTE: This is only important in cells that are actively dividing. Hence, it is not suitable for most neurons.

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Gene “Therapy”(gene transfer)

(1) Insert the gene of interest into a benign viral vector. (Choice ofvector can be important).

(2) Use microinjection to flood a specific area/nucleus of the brain with the viral vector.

(3) Some neurons will take in the virus and incorporate the transgene into the nuclear DNA.

(4) Afterward, assay the area for mRNA activity, proteins, etc. to see the effect of the transgene.

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Gene Therapy

Notes:

Often a marker gene is also included in the viral vector to assess how well the neurons take up the vector (e.g., GFP or green fluorescent protein).

Useful to study overexpression of a gene product. (E.g., overexpression of CREB in hippocampus).