Question 1

Describe the method for creating a transgenic mouse by pronuclear injection. What information can a transgenic mouse provide?

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Question 2

You are trying to make a transgenic mouse but there is very poor survivorship of the embryos after introduction of the foreign gene by pronuclear injection. Describe a method you could use to get around this problem.

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Question 3

You have cloned the cDNA for human XPA. You now attempt to use the human XPA cDNA (linked to a promoter) to correct the genetic defect in mice that have the mouse equivalent of the human disease xeroderma pigmentosum, by making transgenic mice. Briefly explain how you would analyze the transgenic mice for:
Stable integration of the transgene into the mouse's genomic DNA
Transcription of the transgene
Translation of the transgene
Ability of the XPA protein to correct the genetic defect in the transgenic mice.

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Question 4

Describe the method for creating a knockout mouse by gene targeting. Explain the importance of selectable marker genes in this procedure. What information can a knockout mouse provide?

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Question 5

You have created a strain of knockout mice. Describe an experiment to confirm that the mice lack expression of the targeted gene at the level of mRNA expression. Show sample positive results.

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Question 6

Discuss some of the pros and cons of using genetically modified nonhuman primates in experimental medicine.

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Question 7

Describe a recently developed method for creating transgenic livestock by linker-based sperm-mediated gene transfer. Why don't investigators just use pronuclear microinjection?

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Question 8

Discuss some of the potential applications of "gene pharming."

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Question 9

Describe amphibian nuclear transplantation experiments that established the basic principle that cell differentiation depends on changes in gene expression not changes in the content of the genome.

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Question 10

Describe the method for cloning of mammals by nuclear transfer. What information can a cloned organism provide?

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Question 11

Less than 1% of all nuclear transfers from adult differentiated cells result in normal-appearing cloned offspring. Give possible explanations for this lack of efficiency.

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Question 12

Will a cloned calico cat have the exactly the same pattern of orange and black patches on her coat compare with her calico donor? Explain your answer.

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Question 13

Discuss some of the controversial applications of cloning by nuclear transfer.

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Question 14

Describe an experiment to determine whether the tomato you just bought from the grocery store is genetically modified. Show sample positive results.

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Question 15

Design a Southern blot experiment to check a transgenic mouse's DNA for integration of the GFP cDNA under control of a constitutive promoter. You many assume any array of restriction sites you wish in the target mouse chromosome and in the GFP transgene. 
(a) Show sample results for a successful and an unsuccessful integration.
(b) Will mice from different founder lineages have the same site of integration?
(c) Describe techniques you would use to analyze the transgenic mice for transcription of the transgene and translation of the transgene.

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Question 16

What would be the outcome if the procedure used to make gene-targeted (knock-out) mice was altered in the following ways (consider each situation separately). (a) The embryo-derived stem (ES) cells did not possess a gene responsible for visible phenotypic trait (such as agouti coat color) that differed from that of the cell in the recipient blastocyst. (b) The inserted "disrupting" gene (e.g. neo^R) did not insert into the gene of interest. (c) Chemical transfection or electroporation failed. (d) The gene knock-out was embryonic lethal

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Question 17

Consider the following hypothetical scenario:
A young scientific genius, Dolly, is terminally ill with hereditary nonpolyposis colon cancer (HNPCC). When she dies, her parents feel that one of the most remarkable minds in science will die with her, and they feel they owe it to the world to not let this happen. The family travels to a secret lab on a small offshore island, which allegedly performs "reproductive cloning"
(cloning by somatic cell nuclear transfer). Dolly's parents hope to clone her from one of her skin cells.
(a) If the lab is successful in cloning Dolly, can they guarantee that her clone will be as academically gifted as Dolly? Why or why not?
(b) Would Dolly and her clone have identical DNA "fingerprints"? Why or why not?
(c) Would Dolly and her clone share the same mitochondrial DNA? Why or why not?
(d) Would you expect there to be a difference in the length of telomeres in Dolly's skin cells versus in the cells of her embryonic clone? Why or why not?

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A young scientific...

Question 18

A team of molecular biologists seeks your advice regarding whether it would be more important to include regulatory elements such as insulators and matrix attachment regions in a gene construct for generating transgenic mice by gene-targeting in embryo-derived stem cells, or for generating transgenic mice by microinjection of foreign DNA into pronuclei of fertilized eggs? Provide advice and explain your rationale.

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Quiz 15: Genetically Modified Organisms: Use in Basic and Applied Research