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

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Question 1

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

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The method for cloning mammals by nuclear transfer involves taking the nucleus of a somatic cell from the animal to be cloned and transferring it into an enucleated egg cell. The egg cell is then stimulated to divide and develop into an embryo, which is then implanted into a surrogate mother for gestation. This process has been successfully used to clone animals such as Dolly the sheep and has the potential to be used for the conservation of endangered species and for medical research. A cloned organism can provide valuable information about the role of genetics in development and disease. It can also be used to study the effects of environmental factors on gene expression and to produce genetically identical animals for research purposes. Additionally, cloned organisms can be used to create genetically modified animals for the production of pharmaceuticals or for agricultural purposes. Overall, the cloning of mammals by nuclear transfer has the potential to advance our understanding of genetics and to contribute to important scientific and medical advancements.

Question 2

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When making a transgenic mouse by microinjection of foreign DNA into fertilized eggs

A) there is random integration of the transgene into the embryonic genome.
B) there is disruption or mutation of a targeted gene in the embryonic genome.
C) the resulting mice are genetically identical to the donor nucleus.
D) the integration event into the embryonic genome usually occurs by homologousrecombination.

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Question 3

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Plasmids are used as vectors in both plant and bacterial recombinant DNA technology. However, there is a major difference in the fate of genes introduced into bacteria on most bacterial plasmids and into plants on Ti plasmids. What is this difference?

"Pharming" is

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

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

When nuclear transfer is performed by cell fusion, the resulting cloned offspring

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.

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.

Which of the following correctly orders cells according to their phenotypic potential from greatest to least?

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.

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?

Assume you have transfected embryonic stem cells with a gene-targeting vector containing the neomycin resistance gene as a positive selection marker and the thymidine kinase gene as a negative selection marker. You observe that some cells are resistant to neomycin but killed by ganciclovir. What was the outcome of gene targeting procedure?

The term "reprogramming" refers to

Creation of which of the following does not rely on homologous recombination into the genome?

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

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.

You have received a lineage of "knockout mice" from a collaborator. What method would not be useful to confirm that the mice really do not express the targeted gene in all tissues of the body?

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

When making transgenic mice, to determine whether there has been stable integration of a transgene into the mouse chromosome, tail biopsies are taken from the mouse pups and tested for the presence of the transgene by