Genetics - Problem Drill 19: Dissection of Gene Function: Mutational Analysis of Model Organisms

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1 Genetics - Problem Drill 19: Dissection of Gene Function: Mutational Analysis of Model Organisms No. 1 of The mouse gene knockout is based on. (A) Homologous recombination (B) Site-specific recombination (C) Anti-sense technology (D) DNA footprinting (E) DNA double-strand break repair A. Correct! This is the most widely used technique in mouse gene knockout. Homologous recombination is based on gene sequence, but it is not site-specific. Anti-sense technology is used in gene silencing, not gene knockout. DNA footprinting is the sequence that is left around by transposons, not part of gene knockout. DNA double-strand break needs homologous recombination to be repaired but is not the basis for gene knockout. Gene knockout is one of the most powerful techniques generally available to assess gene function in vivo. (A)Homologous recombination

2 No. 2 of The mechanism of RNAi is. (A) Deletion of DNA segments (B) Block translation of mrna (C) Degrade mrna (D) Inhibit transcription of the DNA template (E) Degrade protein RNAi cannot affect gene function at the DNA level. RNAi does not directly block translation. C. Correct! RNAi degrades mrna and, therefore, there will be no protein synthesis. RNAi does not affect how much mrna is synthesized from DNA template; rather, it targets the mature RNAs in cytosol and degrades them. RNAi does not degrade protein. RNAi is widely used in gene silencing experiments. (C)Degrade mrna

3 No. 3 of Which of the following statement is NOT true? (A) RNAi is one approach of reverse (B) When a gene sequence is known, an RNAi or a homologous recombinationbased gene knockout approach can be used to characterize the gene s function in vivo. (C) Transposon-tagged Arabidopsis can be used in both forward and reverse (D) For forward genetics, it is necessary to map the target gene on the chromosome. (E) Homologous recombination is often used in forward This statement is true; RNAi is one approach of reverse This statement is true. RNAi and gene knockout are the two most powerful approaches for analysis of gene function. This statement is also true; transposon tagging can be used to make a mutant line. Additionally, it can also be used in genome-wide tagging and genomics for reverse This is also true; genetic mapping is a necessary step for forward E. Correct! Homologous recombination is most often used in reverse genetics, not in forward The E statement is not true since homologous recombination is more often used in reverse (E)Homologous recombination is often used in forward

4 No. 4 of A chimeric mouse is. (A) A mouse heterozygous for one of its coat color genes. (B) A mouse progeny from the cross of two different lines. (C) A mouse with sections developed from two different types of embryonic cells. (D) A mouse with xenografted tissues. (E) A mouse with tumors. A gene knockout mouse may have a chimeric phenotype coat color, which is a designated selection marker for knockout gene, but the color itself is not how a chimeric mouse is defined. A mouse progeny from a cross of two different lines is called a hybrid mouse, not a chimeric mouse. C. Correct! This is the first mouse obtained that may carry a knockout construct. This is not a chimeric mouse; rather, it is a mouse with extra tissues. A mouse with a tumor is a mouse whose genes may have mutations in tumor suppressors or oncogenes, not a chimeric mouse. A chimeric mouse is the first mouse that is likely to carry a disrupted gene. (C)A mouse with sections developed from two different types of embryonic cells.

5 No. 5 of Which of these steps is NOT involved in forward genetics? (A) Chemical mutagenesis. (B) Transposon tagging. (C) Genetic mapping. (D) Isolation of the transposon-flanking sequence. (E) Making a knockout construct. Chemical mutagenesis is a common method to generate mutants in forward Transposon tagging is another common method to generate mutants in forward Genetic mapping is a necessary step in forward Isolation of the transposon-flanking sequence is a step for isolating the target gene. E. Correct! Making a knockout construct is not needed in forward genetics; it is part of reverse This problem outlined all steps involved in forward (E)Making a knockout construct.

6 No. 6 of Which of the following statements is a benefit for a model organism used in genetic studies? (A) A random, unknown genetic background. (B) A well-established genetic background. (C) An uncontrolled mating pattern. (D) A long, healthy life cycle. (E) Only a small number of offspring of one or two. A well-established genetic background is beneficial for a model organism. B. Correct! A well-established genetic background is beneficial for a model organism. Controlled mating is required. A relatively short life cycle is required, so the experiments can be done in a reasonable time frame. A relatively large number of offspring is better. There are certain characteristics that make an organism a candidate for the role of model organism. These characteristics include: Amenability to experimental manipulation, well-established genetic background, relatively short life cycle, relatively large number of offspring from a mating, controlled mating, and genetic variations. (B)A well-established genetic background.

7 No. 7 of Which of the following statements about producing a mutant for genetic studies is correct? (A) Two methods of mutagenesis for mutant identification studies include transposon tagging and transposition. (B) In transposon tagging, the mutation rate is very high. (C) The mutant allele in transposon tagging can t be used for gene isolation. (D) Chemical mutagenesis has a low mutation rate. (E) The mutation rate during chemical mutagenesis is high. Two methods are transposon tagging and chemical mutagenesis. Transposon tagging has a relatively low mutation rate. In transposon tagging, the mutant allele can be used later for gene isolation. Chemical mutagenesis has a high mutation rate. E. Correct! Chemical mutagenesis has a high mutation rate. Transposon Tagging: The mutation rate is relatively low and the mutant allele can be used later for gene isolation. Chemical Mutagenesis: the mutation rate is high and the mutant allele cannot be used for gene isolation. (E)The mutation rate during chemical mutagenesis is high.

8 No. 8 of Gene isolation. (A) Involves separation by electrophoresis of mutant and wild type samples. (B) In transposon tagging, electrophoresis is used to separate only the mutant sample. (C) Involves the use of no specific probe, which is needed to identify the gene. (D) Studies would involve sequencing the entire DNA because the mutant and wild type will migrate identically. (E) Can t be done with transposon tagging. A. Correct! Gel electrophoresis is used for the separation of wild type and mutant samples for mutant allele tagging studies. Gel electrophoresis is used to separate the mutant and the wild type samples to compare the results. The mutant is used as a probe to detect the target DNA sequence. The DNA band that co-migrates with the mutant allele is isolated and sequenced. The mutant allele can be used for gene isolation studies. DNA from mutant plants and sibling wild type plants are isolated and their DNA is digested and separated by gel electrophoresis. Transposon DNA is used as a probe to detect the target DNA sequence. The DNA band that co-migrates with the mutant allele is isolated and sequenced. (A)Involves separation by electrophoresis of mutant and wild type samples.

9 No. 9 of Which of these steps about embryonic stem cells is correct? (A) Embryonic stem cells come from the bone marrow. (B) Human embryos reach the blastocyst stage around 5 months after fertilization. (C) Embryonic stem cells can differentiate into any cell type in the bloodstream. (D) Both embryonic stem cells and multipotent stem cells found in adults can form any cell type in the body. (E) Multipotent stem cells found in adults can form different cell types but not as many as embryonic stem cells. Embryonic stem cells come from the blastocyst. The blastocyst stage is around 5 days after fertilization. Embryonic stem cells can differentiate into all the different cell types of the body. Multipotent stem cells can form different cell types in the body, while embryonic stem cells can form all cells of the body. E. Correct! Multipotent stem cells can form different cell types in the body, while embryonic stem cells can form all cells of the body. Embryonic stem cells (ES cells) come from the blastocyst. Human embryos reach the blastocyst stage around 5 days after fertilization. ES cells are pluripotent, meaning they can differentiate into all cell types of the adult body. Pluripotent is different then multipotent found in adults, which can form only limited types of cells. (E)Multipotent stem cells found in adults can form different cell types but not as many as embryonic stem cells.

10 No. 10 of Which of the following statements about foreign DNA and embryonic stem cells is true? (A) The only method to get foreign DNA into embryonic stem cells is through the use of viral vectors. (B) Foreign DNA can be introduced into embryonic stem cells using techniques, such as: electroporation, microprecipitates, and viral vectors. (C) The embryonic cells are screened for DNA uptake using modified medium conditions containing phages. (D) When potentially successful embryonic stem cells are grown in the appropriate antibiotic containing medium, all cells in the culture will die. (E) Embryonic stem cells that have successfully taken up the foreign DNA will be rendered more susceptible to the antibiotic used during cell screening. Foreign DNA can be introduced into embryonic stem cells using: electroporation, microprecipitates, viral vectors, microbombardment, liposomes, and microinjection. B. Correct! Foreign DNA can be introduced into embryonic stem cells using: electroporation, microprecipitates, viral vectors, microbombardment, liposomes, and microinjection. Modified medium containing an antibiotic, such as neomycin, is used to screen for those embryonic stem cells that successfully took up the foreign DNA containing the antibiotic-resistant gene. If successfully taken up, the foreign DNA will facilitate antibiotic resistance and those cells will not die. The foreign DNA will render the cells antibiotic-resistant. There are several methods used to get DNA into ES cells. These methods include: electroporation, microprecipitates, microinjection, virus vector, liposomes and microbombardment. Neomycin is added to the growth media, which will ensure that only cells having the integrated construct will grow (positive selection). ES clones are cultured and PCR is used to sequence and, thereby, confirm the true homologous recombinants. The ES clones are grown and introduced into a mouse embryo. (B)Foreign DNA can be introduced into embryonic stem cells using techniques, such as: electroporation, microprecipitates, and viral vectors.

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