Chapter 18 Practice Problems Flashcards
gene transferred by a scientist into an organism’s genome
transgene
method of DNA transfer used for many vertebrates
pronuclear injection
vector for making conditional knockouts
T-DNA
genetically engineered viral genome that transfers a therapy gene
AAV vector
generate viral particles for gene therapy
packaging cells
endonuclease used for genome editing along with sgRNA
Cas9
loss of function mutant through gene targeting
knockout mouse
contains additional or altered DNA through gene targeting
knockin mouse
causes crossover at loxP sites
Cre recombinance
can develop into any cell tyep
ES cells
plant or animal that carries a transgene
GM organism
sometimes, genes transferred into the mouse genome by pronuclear injection disrupt a gene at the random site of integration, resulting in a mutation. In one such case, investigators identified a recessive mutation that causes limb deformity in transgenic mice.
a. the mutant in this example was in fact caused by the insertion of the transgene. How could you use this transgene insertion as a tag for identifying the mutant gene responsible for the aberrant limb phenotype?
b. the mutation in this example was in fact caused by the insertion of the transgene. How could you use this transgene insertion as a tag for identifying the mutant gene responsible for the aberrant limb phenotype?
c. the insertion mutation was mapped to chromosome 2 of mice in a region where a recessive mutation called limb deformity had been identified previously. Mice carrying this mutation are available from a major mouse research laboratory. How could you tell if the ld mutation was in the same gene as the transgene insertion mutation?
a. Cross the transgenic strain to wild type, and cross F1 animals heterozygous for the transgene insertion to each other. If limb deformity is not caused by the transgene insertion, some F2 animals homozygous for the transgene insertion will not have the mutant phenotype. If the limb deformity is due to the transgene insertion, homozygosity for the transgene should correlate with limb deformity.
b. inverse PCR
c. conduct a complementation test
The fly eyes are malformed because they lack a functional copy of a gene called fat facets that is required for eye development. The human genome encodes a protein called Usp9 that is similar in amino acid sequence to the fly Fat facets protein. Likewise, the mouse genome encodes a protein called Fam that is similar to Fat and Usp9
a. How could you determine if human Usp9 can substitute for the drosophila Fat protein
b. how could you determine if, like Fat in flies, the mouse Fam protein is required for mouse eye development?
a. Transform faf- flies with a construct in which the faf gene regulatory sequences from Drosophila drive expression of a Usp9 cDNA. If the rough eye phenotype is rescued to wild-type, the human USP9 protein can in fact substitute functionally for fly Faf protein.
b. If mice with loss-of-function Fam- mutations have mutant eyes, then Fam must be required for mouse eye development. In Section 18.3, you will learn how to use cloned Fam DNA to generate mice with loss-of-function Fam- mutants; these will be knockouts or conditional knockouts.
6. a. Fly lines in which the enhancer trap construct integrated next
a. describe two ways you could potentially make a transgene that would inhibit the function of a specific gene in a transgenic organism
b. discuss how you could use either of these methods to construct a mouse model for a recessive human genetic condition associated with a loss of function such as cystic fibrosis
a. to construct a transgene that expresses a dominant negative allele of the gene.
b. An RNAi construct for the CFTR gene could be made that consists of one or a few of the large number of exons of the gene, and two copies of a strong lung-specific promoter oriented as shown in the answer to part (a). The transgene could be incorporated into the mouse genome by pronuclear injection.For the CFTR gene, it also may be possible to generate a dominant negative by using a very strong lung promoter to overexpress an allele encoding a mutant protein that cannot function as a chloride ion regulator (due to a deletion in the ORF or a missense mutation), but that can insert into the cell membrane. As above, the transgene would be transferred to the mouse genome by pronuclear injection.
