CHE391L/S13/Genome Editing: Difference between revisions
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==Introduction== | ==Introduction== | ||
The ability to alter the genomes of living organisms has been critical to our understanding of genetics and the development of synthetic biology as a viable field. In it's simplest form, genome editing involves generation of gene knockouts, where expression is eliminated through insertion or a removal of a region of genomic DNA, or knockins, where a new coding region is inserted to produce a novel gene product. This process is fairly straightforward in bacteria and yeast, where a cell's own homologous recombination machinery can be used to make genomic insertions, albeit with low efficiency. To carry this out, the cells are transformed with exogenous DNA (usually on a plasmid) containing the desired insertion sequence flanked by homology regions complementary to the target site sequences. Because very few cells undergo successful recombination, the inserted sequence must contain a selectable marker, such as an antibiotic resistance gene, to facilitate selection of modified cells. Thus, most knockouts are generated simply by inserting a marker in place of an existing gene, thus eliminating its expression. | |||
Genomic insertions can also be generated in higher eukaryotes using homologous recombination, but the process is significantly more involved. Knockout mice have been a staple of genetics research since the 1980s, but they can take upwards of a year to generate. The process begins with embryonic stem cells (ESCs) harvested from a mouse blastocyst. They are then transfected with insert DNA by electroporation and successfully recombined cells are selected using an antibiotic such as neomycin. The surviving ESCs are then injected into another blastocyst and implanted into a surrogate mouse's uterus. Some of the resulting pups will be chimeric animals with a portion of their cells containing the modification. Subsequent breeding of the chimeras allows for generation of a knockout animal. | |||
Revision as of 06:20, 8 April 2013
Introduction
The ability to alter the genomes of living organisms has been critical to our understanding of genetics and the development of synthetic biology as a viable field. In it's simplest form, genome editing involves generation of gene knockouts, where expression is eliminated through insertion or a removal of a region of genomic DNA, or knockins, where a new coding region is inserted to produce a novel gene product. This process is fairly straightforward in bacteria and yeast, where a cell's own homologous recombination machinery can be used to make genomic insertions, albeit with low efficiency. To carry this out, the cells are transformed with exogenous DNA (usually on a plasmid) containing the desired insertion sequence flanked by homology regions complementary to the target site sequences. Because very few cells undergo successful recombination, the inserted sequence must contain a selectable marker, such as an antibiotic resistance gene, to facilitate selection of modified cells. Thus, most knockouts are generated simply by inserting a marker in place of an existing gene, thus eliminating its expression.
Genomic insertions can also be generated in higher eukaryotes using homologous recombination, but the process is significantly more involved. Knockout mice have been a staple of genetics research since the 1980s, but they can take upwards of a year to generate. The process begins with embryonic stem cells (ESCs) harvested from a mouse blastocyst. They are then transfected with insert DNA by electroporation and successfully recombined cells are selected using an antibiotic such as neomycin. The surviving ESCs are then injected into another blastocyst and implanted into a surrogate mouse's uterus. Some of the resulting pups will be chimeric animals with a portion of their cells containing the modification. Subsequent breeding of the chimeras allows for generation of a knockout animal.
