Altering the Genome by Gene Targeting




Isolation of embryo-derived stem (Es) cells from mouse blastocysts. a 4.5 day preimplantation embryo, the blastocyst, is placed on a monolayer of cells which provide a matrix for attachment as well as secreted protein factor(s) whose role is to inhibit newly born Es cells from differentiation. following attachment of the blastocyst, the cells in the inner cell mass proliferate. at the appropriate time the cell mass is physically removed with a micropipette, dispersed unto small clumps of cells. and seeded onto new feeder cells. colonies are microscopically examined for the characteristic morphology of Es cells. such colonies are picked, dispersed in to single cells and reseeded onto feeder cells. if the procedure progresses successfully these cells yield colonies with a uniform morphology, characteristic of Es cells. the Es cells can then be tested for their capacity to differentiate in vitro as well as their capacity to generate germ-line chimeras.

How to replace a targeted gene in cultured cells.

After you have your cultured cells from the previous procedure.
1. create a targeting vector and insert a neomycin resistance (neo r)gene into a protein coding area. This will act as a marker when the mutated cell is exposed to neomycin, cells that have the neo r gene will be immune to the antibiotic. The vector is also attached to a herpes tk gene. 2. vectors are introduced into prepared cells
3. Homologous recombination occurs when the targeted gene lines up with the normal gene, and replaces the normal code with the altered one, including the information between (the neo r gene) but not the tk gene. The most often mistake is when the entire vector randomly inserts itself into a chromosome, however when this occurs the herpes tk gene will also be included.





To determine which cells have the targeted mutation, they are transferred into a medium containing neomycin, and ganciclovir, a drug that kills cells that have a herpes tk gene. Cells with out the neo r gene will die upon exposure to neomycin. This will leave only cells with the proper insertion vector. This unfortunatly occurs only 1 in a million trys. most times the vector fails to intergrate at all.

How Targeted Gene Relacement Works in Mice




1. Take our cultured Es cells and altered with a vector. They use coat color as an indicator, by placing the cells into a blastocyst stage embryo, which is grown in a surrogate mother, that otherwise would have a completely black coat. The single copy of the agouti gene will cause a change in coat color in those mice with the desired mutation.

2. The mice are separated by coat color,at around 3 weeks of age, chimeras (when cells are derived from two different sources) will have a brownish coat, indicating acceptance of the vector.

3. these mice are then mated with black mice, their offspring will then be separated by coat color and then their genes will be examined directly for the mutation.

4. these animals are then mated to each other to create homozygous for the targeted gene mice. These mice have their genes screened directly and are checked for abnormalities. This is how the function of the knocked out gene is determined.

Homeotic Genes



Homeotic or Hox genes are responsible for ensuring the parts of the body differentiate into the appropriate organs, and have the proper shape.
Drosophila have 8 Hox genes where humans and mice have 38. The assumption is the extra Hox genes are needed for the more complex mammalian body. And were crucial during the evolution from invertebrates to vertebrates
The human disease Di George syndrome is similar to the effects that a defective HoxA-3 gene in mice creates. The HoxA-3 gene controls tissue development that occurs in the tissues that descend from a particular group of embryonic cells. Homozygous mice for the mutated copy die at birth from cardiovascular dysfunction caused by malformed heart vessels and muscle. Di George syndrome is not cause by a bad HoxA-3 gene in humans however it is thought to be caused by a gene that interferes with HoxA-3.

Why Should We Care

Using the mouse model for Di George is now available and will help develop clues to treatment.
Immunologists have also benefited from gene targeting in determining the development of b and t lyphoctes.
Cancer Researchers are using the Knockout technology to tumors and mutations of the p53 suppresser gene. Over 80% of the time human cancers have a mutated p53 gene. The loss of the p53 gene allows damaged DNA to be passed on and this leads to eventual cancer growth.

The order of the Hox genes on the chromosomes is the same in Drosophila, mice and humans. These genes are distributed in 4 different linkage groups on four separate chromosomes. Because the Hox genes are so critical to the placement and formation of body tissues mutations in the Ox genes cause defects in all body systems from the brain, to the heart, to the skeletal system.


Gene targeting can also be expected to contribute significantly to neurobiology, providing a rational and new approach to genetic analysis of the intimidatingly complex mammalian nervous system. As a representative mammal the mouse should again profit from the molecular-genetic analysis of less Complex organisms such as Drosophila. Many interesting genes that affect the functioning of the nervous system in these organisms are likely to he conserved in mammals. Gene targeting should allow the evaluation of the roles of such genes as well as the rotes of genes recently identified directly in the mouse on the basis of intriguing, highly neuron-specific patterns of expression. By combining this new genetic analysis with other already established analytical approaches, we might begin to achieve some understanding of a molecular mechanism underlying development of the mammalian nervous system as well as its most elusive functions such as learning and memory.

Of more immediate application to human medicine, targeted disruptions in the mouse should provide mouse models for human genetic diseases. Such models should prove useful for analyzing the pathology of the disease as well as providing systems for exploration of new therapeutic protocols, including gene therapy. It will be interesting to see if gene targeting in human somatic cells can be directly applied to human gene therapy. Correction of the endogenous defective gene in the appropriate human tissue has obvious advantages over the random insertion of a nondefective gene: for example, the corrected endogenous gene is much more likely to be expressed at appropriate levels. As researchers become able to culture and propagate a variety of human somatic stem cells such as hematopoietic stem cells, epithelial stem cells, liver and lung stem cells, somatic gene therapy protocols may become possible based on gene targeting to correct the endogenous gene in the appropriate tissue.





I drew heavily on Sci Am March '94 and TIG vol 5, no 3
Bibliographies can be found on our sources page

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