Passage Overexpression of the miR-17~92 gene cluster occurs in many human cancers involving c-Myc, a gene coding for a transcription factor that regulates expression of proteins such as p21, an inhibitor of cell cycle progression. miR-17~92 encodes six distinct microRNAs (miRNAs) that can be divided into four families based on sequence identity at positions 2-7 (Table 1).Table 1 Sequence Identities of miRNA Families Encoded by the mir-17∼92 Gene Cluster Experiment 1Two independent, diffuse B-cell lymphoma cell lines were used to study the functional relationship between miR-17~92 transcripts and c-Myc in cancer pathogenesis. The first cell line was derived from Eμ-Myc mice (Eμ), which overexpress a c-Myc transgene under the control of a B-cell-specific enhancer that induces early development of B-cell lymphoma. The second cell line was derived from Eμ-Myc mice carrying a miR-17∼92 knockout allele (Δ/Δ). As shown in Figure 1, researchers measured the growth of both cell lines, as well as that of Δ/Δ cells infected with a retrovirus expressing the entire miR-17∼92 cluster (Δ/Δ + miR-17∼92). Figure 1 Growth curves of Eμ cells, Δ/Δ cells, and Δ/Δ + miR-17∼92 cells (error bars = standard deviation)Experiment 2To assess the relative contribution of each miRNA to the oncogenic ability of the miR-17∼92 cluster, Δ/Δ cell lines were transduced with an empty vector or with miR-17∼92 mutant allele constructs that were engineered to lack expression of miRNA from at least one of the four families. In each of the constructs, the family that was knocked out of the vector is indicated by the Δ symbol. After verifying that the transduced cells correctly expressed the desired miRNA, the fraction of apoptotic cells in each line was measured (Figure 2). Figure 2 Percentage of apoptotic cells in Eμ cells and Δ/Δ cells transduced with the indicated miR-17~92 construct (error bars = standard deviation) Adapted from Mu P, Han YC, Betel D, et al. Genetic dissection of the miR-17~92 cluster of microRNAs in Myc-induced B-cell lymphomas. Genes Dev. 2009;23(24):2806-11. -As described in the passage, verification that the transduced cells expressed the desired miRNA was an essential experimental step to ensure that:

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Quizplus · Accepted Answer

11ecba2d_0f62_0807_a3bf_6b8bc04fe053_MD0008_00 Positive controls are often employed to ascertain test validity and allow researchers to correctly interpret scientific data. As part of proper experimental design, positive controls allow investigators ensure that the dependent variable to be studied is present or that the treatment meant to manipulate the dependent variable functions properly. In Experiment 2, verifying that each mutant construct works properly and expresses only the desired families serves as a positive experimental control. In Figure 2, the independent variable (treatment) is the type of vector transduced into Eμ-Myc cells with miR-17∼92 knocked out (Δ/Δ), and the dependent variable is the percentage of apoptotic cells. The purpose of measuring apoptosis under these conditions was to determine precisely which miRNA families contribute the most to miR-17~92 function. To reliably draw such conclusions, it was necessary to first ensure that deletion of one or more miRNAs encoded by the miR-17∼92 cluster did not affect expression/processing of the remaining miRNAs encoded by the mutant constructs. (Choice A) Although researchers assessed the fraction (percentage) of apoptotic cells in samples with different genotypes, the rate of apoptosis (ie, how fast cells died) was not measured. (Choice B) According to the passage, all of the cell lines tested were derived from Eμ-Myc mice, which already exhibited early development of diffuse B-cell lymphoma. (Choice D) The purpose of the experiment was to determine the extent to which introduction of constructs containing one or more miR-17∼92 alleles did promote cell growth in the lines tested. Educational objective: Positive controls are often employed to ascertain test validity and allow researchers to correctly interpret scientific data. This kind of experimental setup can verify that the dependent variable to be studied is present or that the treatment (independent variable) meant to manipulate the dependent variable functions properly.

Passage Overexpression of the MiR-17~92 Gene Cluster Occurs in Many Human