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:

A) lack of expression of one or more miRNA encoded by the miR-17∼92 cluster did not affect the rate of apoptosis in tested cells. B) transduction of tested cells with mutant miR-17∼92 alleles did not induce the development of B-cell lymphoma. C) deletion of one or more miRNA encoded by the miR-17∼92 cluster did not affect expression of the remaining miRNAs encoded by the constructs. D) expression of one or more mutant miR-17∼92 alleles did not provide the transduced cells with a selective growth advantage. A) lack of expression of one or more miRNA encoded by the miR-17∼92 cluster did not affect the rate of apoptosis in tested cells. B) transduction of tested cells with mutant miR-17∼92 alleles did not induce the development of B-cell lymphoma. C) deletion of one or more miRNA encoded by the miR-17∼92 cluster did not affect expression of the remaining miRNAs encoded by the constructs. D) expression of one or more mutant miR-17∼92 alleles did not provide the transduced cells with a selective growth advantage.

Passage The nicotinic acetylcholine receptor (nAChR) is a protein embedded in the muscle fiber membrane that facilitates cell-to-cell communication across the neuromuscular junction. When acetylcholine (ACh) binds nAChR, electrical depolarization within skeletal muscle fibers is induced and ultimately gives rise to contractile activity. The nAChR is composed of five protein subunits symmetrically organized around a central ion pore. Previous studies demonstrated that these subunits may serve as epitopes (antibody binding sites) in autoimmune disease. By reversibly binding nAChR subunits, anti-nAChr antibodies may temporarily block the attachment site for ACh, resulting in muscle weakness.Researchers harvested muscle samples from mice to investigate the effects of anti-nAChR antibodies on muscle tension, the pulling force transmitted by muscles during contraction.Experiment 1First, skeletal muscle samples (tibialis anterior) of adult male mice were surgically harvested (n = 30), washed, and stripped of connective tissues. Next, the isolated muscle samples were placed in a 150-mL oxygen-infused, electrolyte-rich bath containing glucose and CaS1U1P12+S1S1P0 ions that were able to enter the muscle fibers and enable maximal contractile force. One end of the submerged muscle was anchored in place while the other was attached to an apparatus that measures muscle tension and modifies muscle length. Using electrodes, action potentials were elicited in the harvested muscle tissue to evaluate contractile function (Figure 1). Figure 1 Action potential elicited from a harvested skeletal muscle sampleExperiment 2The researchers divided the 30 muscle samples into three groups of 10. Resting muscle tension was measured, and the electrolyte-rich baths of each group were further infused with a 7.5-mL solution containing mouse-specific anti-nAChR antibodies, ACh, or a mixture of both (Table 1). Post-infusion (peak) muscle tension was measured for 30 minutes, with results displayed in Figure 2.Table 1 Solutions Infused into the Electrolyte-Rich Bath Figure 2 Pre-infusion resting muscle tension and post-infusion peak muscle tension in harvested muscle samples (Note: * indicates p < 0.05.) -In humans, skeletal muscles are innervated by the somatic nervous system and function to mediate:

A) blood pressure regulation. B) heart contraction. C) propulsion of food contents along the digestive tract. D) movement of the jaw during chewing. A) blood pressure regulation. B) heart contraction. C) propulsion of food contents along the digestive tract. D) movement of the jaw during chewing.