Chapter 17.3: Targeted Mutagenesis

Question 1

Describe construction of knockout mice

Answer 1

 Using recombinant DNA techniques, a gene specifying resistance to the drug neomycin is inserted into an exon of the gene of interest
 Purebred agouti mice are mated to produce an early embryo (a blastocyst). Embryonic stem (ES) cells from the inner cell mass of the blastocyst are cultured to increase their number
 Cultured ES cells are treated with the exon disrupted by neomycin. In a small number of these cells, homologous recombination swaps a wild-type exon on a mouse chromosome in the ES cells with the disrupted version of the exon (a knockout allele). The cells with knockouts are selected by exposing the ES culture to neomycin, and they are grown into colonies.
 Individual neomycin-resistant cell clones are tested to identify a clone where integration occurred by homologous recombination.
 Purebred black mice are mated to produce a blastocyst that is removed from the female.
 About 10 ES cells with the knockout allele from the same colony are injected into the blastocyst from the black mice. This blastocyst is then placed in the uterus of another black female, where it can develop into a live-born mouse.
 The mice that are born are called chimeras, meaning that they are made up of cells from two different organisms: The cells in these animals derived from ES cells are heterozygous for a mutation in the targeted gene, while the host cells are homozygous for wild-type alleles of this locus.
• Geneticists mate the chimeric mice to wild-type mice to generate nonmosaic heterozygous progeny.
o Progeny are called knockout mice because they contain a chromosome with amorphic (knock out) allele of the targeted gene
• The geneticists then cross heterozygous knockout mice to each other to generate homozygous mutants

Question 2

Define gene targeting

Answer 2

o Gene transfer by means of homologous recombination can make mutations in specific bacterial genes

Question 3

Define TALEN

Answer 3

Transcription Activator-Like Effector Nuclease

Question 4

How do you generate single genomes that can be packaged into individual phage particles?

Answer 4

 To generate single genomes that can be packaged into individual phage particles, a protein called Cre recombinase causes crossing-over to occur between two specific DNA sequences on the large circles called loxP sites

Question 5

Why are knockout mice called that?

Answer 5

o Progeny are called knockout mice because they contain a chromosome with amorphic (knock out) allele of the targeted gene

Question 6

What are the benefits of knockin mice?

Answer 6

o Knockin mice homozygous for the mutant gene will produce only the mutant form of the protein, although mice heterozygous for the knocked in mutation may also be valuable if the mutation has dominant effects

Question 7

What means to retain the ability to become any cell type—including germ-line cells and gametes—when exposed to the appropriate signals in a developing embryo?

Answer 7

totipotent

Question 8

What are conditional knockout mice?

Answer 8

o Mice with genes that can be inactivated specifically when investigators alter the environmental conditions

Question 9

Define totipotent

Answer 9

retain the ability to become any cell type—including germ-line cells and gametes—when exposed to the appropriate signals in a developing embryo

Question 10

What is o Gene transfer by means of homologous recombination can make mutations in specific bacterial genes?

Answer 10

gene targeting