Biology 340 Molecular BiologyFeb. 19 and 21, 2001
Lectures 13 and 14
Transgenic and Knockout Mice
Readings: Chap. 8 pp. 281- 289
Sauer, B. (1998) Inducible Gene Targeting in Mice Using the Cre/lox System. Methods: A Companion to Methods in Enzymology 14, 381-392.
Outline:
1. Transgenic vs. knockout mice
2. Transgenic technology
3. ES cells and knockouts
4. Inducible knockouts
Lecture
1. Transgenic vs. knockout mice
An important application of recombinant DNA technology is the production of plants and animals expressing a particular gene of interest. Transgenic animals and plants contain a particular foreign gene, usually inserted in the genome of the host organism.
In other studies, it would be useful to disrupt a particular gene to determine its phenotypic effect. In mice this is accomplished by creating "knockouts", non-functional versions of a gene which are targeted to replace the normal gene.
2. Transgenic technology
Mice can be rapidly produced which contain a particular gene inserted at random into the mouse genome. The technology to produce a transgenic mouse is described in Fig. 8-36 and on the CD-ROM animation, creating a transgenic mouse.
Key steps:
1. Foreign DNA is injected into one of the pronuclei of a fertilized mouse egg prior to fusion of male and female pronuclei.
2. The injected eggs are transferred into a foster mother of a different mouse strain (allows you to tell the transgenic mice apart because they are a different color).
3. About 10 to 30% of offspring contain foreign DNA. This is tested by Southern blots of DNA isolated from mouse tails.
4. Breed transgenic mice to produce pure transgenic strains homozygous for the transgene.
Applications:
--Transgenic mice carrying the "myc" oncogene develop tumors at a high frequency. Used as an animal model for cancer.
--Applying transgenic technology to other animals (goats, pigs, etc.) enables the animals to produce useful products such as proteins or pharmaceuticals.
Knock-out mice: Mice in which the normal germ line copies of genes have been replaced with defective copies (usually deletions). Used to determine the phenotypic effects of genes. Animals in which the allele has been disrupted by a large deletion are often referred to as nulls, alleles that express no functional protein from the altered gene.
Some Applications:
1. Genetic diseases: mdx mouse, a mouse model for muscular dystrophy.
2. Cancer: p53 knockout mice and other knockouts of tumor suppressor genes and oncogenes are models for cancer.
3. Development: mouse knockouts of homeobox genes lead to developmental mutations that parallel other developmental mutants of fruit flies.
Procedures for producing knock-outs:
1. Mutant alleles are introduced by homologous recombination (Fig. 8-33) into embryonic stem (ES) cells. ES cells are totipotent cells capable of differentiating into a wide variety of cell types.
2. ES cells containing one disrupted allele of a gene are injected into early mouse embryos (Fig. 8-34). The resulting mice are chimeras, containing tissues derived from the transplanted ES cells and the host cells.
3. Chimeric mice are mated to produce mice in which the mutant allele is incorporated in the germ line.
4. Mice heterozygous for the knockout mutation are mated to produce homozygous knockout mice.
Key technical step: Engineering the knockout vector to disrupt the gene and select for the mutant allele.
See Fig. 8-33
--Disruption of the gene is a low frequency event, so genetic selection is required.
--Replacement vector contains two dominant selectable marker genes:
neor= neomycin resistance gene, confers resistence to G418 (neomycin)
tk= thymidine kinase gene, makes cells sensitive to the drug ganciclovir, a nucleotide analog
--The mutant allele carries the neor gene; when this allele recombines with the host gene, it can be identified by positive selection for neomycin resistance.
--Non-homologous recombination of the mutant target gene can be counter-selected with ganciclovir; only non-homologous recombinants would retain the tk gene from the construct, while the desired homologous recombinants would lose the tk gene.
Another technical concern in construction of knockouts: Growth of ES cells and insertion of genes:
1. ES cells are derived from mouse embryos; they are not from established cell lines, thus are technically difficult to grow.
2. ES cells are subcultured on a layer of feeder cells, cells that assist the ES cells to attach to a surface and also help provide growth factors and metabolites.
3. It is technically difficult to introduce foreign genes into ES cells; typically a method called electroporation is used in which an electrical shock changes the properties of the cell membranes so they can take up foreign DNA. Transfection (the introduction of DNA) of ES cells by electroporation may be only 5% efficient.
Inducible knockouts: The Cre/lox system
Disruption of many essential genes is lethal to mice. To study the effects of knockout genes without the problem of lethality, a clever genetic control system (Cre/lox) has been devised that allows for targeted disruption of genes only in a particular cell or tissue type.
loxP: site specific recombination sites.
Cre: enzyme that catalyzes recombination between loxP sites
These are derived from E. coli bacteriophage P1 (a circular lysogenic virus).
1. To engineer the knockout, loxP sites are inserted so they flank the gene of interest (Fig. 8-35) and introduced into one mouse strain.
2. Transgenic mice are also prepared that carry the Cre gene linked to a cell-type specific promoter (region on DNA that controls transcription).
3. Mating of the two strains of mice gives progeny that carry the loxP sites flanking the gene of interest and the Cre gene controlled by a cell-type specific promoter. Recombination will only occur in the cells where the promoter is active, so the gene is only knocked out in certain tissues.
4. The mice containing the loxP mutant allele can be crossed with different mouse strains with Cre under control of different tissue-specific promoters to look at phenotypic effects in different tissues.
Cre recombinase system:
--38 kDa recombination enzyme of E. coli P1 virus
--requires 34 bp site called loxP (locus of X-over of P1)
--34 bp loxP site consists of 2 13-bp inverted repeats flanking an 8 bp nonpalindromic core region.
--presence of 2 directly repeated loxP sites allows homolologous recombination to remove a covalently closed circle (Fig. 1 of Sauer).
--a single loxP site remains after recombination
Example: Cre/lox knockout of the NMDA receptor
--neonatal lethal
--Use Cre gene controlled by hippocampal neuron specific promoter to activate targeted knockout of NMDA receptor subunit gene with loxP sites.
--Knockouts survive to adulthood and show learning and memory defects.
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