Passage
In the central nervous system, myelin produced by oligodendrocytes (glial cells) functions as an insulating sheath surrounding certain nerve fibers. To identify candidate genes involved in the myelination process, researchers collected oligodendrocytes from zebrafish and purified the messenger RNA (mRNA) transcripts expressed by these glial cells. The mRNA was converted to complementary DNA (cDNA) by reverse transcriptase, and the cDNA was fluorescently labeled and assessed for hybridization on a microarray. The expression of mRNA in other zebrafish cells was similarly measured.Transcripts detected in zebrafish oligodendrocytes at levels greater than 3 times those found in other cells were selected as candidate myelination genes. Some of the detected genes, including plp1a, Sox10, and mbp, were previously known to be specific to oligodendrocytes, validating the procedure. The cDNA of newly identified candidates was amplified by PCR, and the ends were digested with the restriction enzymes EcoRI and XhoI. Genes were then ligated into the multiple cloning site (MCS) of the pSKII vector, shown in Figures 1 and 2, which had also been digested by EcoRI and XhoI.
Figure 1 Overview of the pSKII vector (plasmid)
Figure 2 The pSKII MCS, showing the restriction sites of several restriction enzymesTable 1 shows the functional parameters of several restriction enzymes.
To confirm that the candidate genes originated in oligodendrocytes, complementary RNA (cRNA) strands were synthesized from the cloned plasmids and hybridized to fixed zebrafish sections. One of the genes that localized to oligodendrocytes, known as cldnk, produces a 915 base pair mRNA transcript from two exons. The cldnk gene is involved in tight junction formation and may be required for myelin sheath development around axons.
-Which experiment could confirm that the cldnk gene product is required for myelination?

Question

Passage
In the central nervous system, myelin produced by oligodendrocytes (glial cells) functions as an insulating sheath surrounding certain nerve fibers. To identify candidate genes involved in the myelination process, researchers collected oligodendrocytes from zebrafish and purified the messenger RNA (mRNA) transcripts expressed by these glial cells. The mRNA was converted to complementary DNA (cDNA) by reverse transcriptase, and the cDNA was fluorescently labeled and assessed for hybridization on a microarray. The expression of mRNA in other zebrafish cells was similarly measured.Transcripts detected in zebrafish oligodendrocytes at levels greater than 3 times those found in other cells were selected as candidate myelination genes. Some of the detected genes, including plp1a, Sox10, and mbp, were previously known to be specific to oligodendrocytes, validating the procedure. The cDNA of newly identified candidates was amplified by PCR, and the ends were digested with the restriction enzymes EcoRI and XhoI. Genes were then ligated into the multiple cloning site (MCS) of the pSKII vector, shown in Figures 1 and 2, which had also been digested by EcoRI and XhoI.

Figure 1 Overview of the pSKII vector (plasmid)

Figure 2 The pSKII MCS, showing the restriction sites of several restriction enzymesTable 1 shows the functional parameters of several restriction enzymes.

To confirm that the candidate genes originated in oligodendrocytes, complementary RNA (cRNA) strands were synthesized from the cloned plasmids and hybridized to fixed zebrafish sections. One of the genes that localized to oligodendrocytes, known as cldnk, produces a 915 base pair mRNA transcript from two exons. The cldnk gene is involved in tight junction formation and may be required for myelin sheath development around axons.
-Which experiment could confirm that the cldnk gene product is required for myelination?

Quizplus · Accepted Answer

11ecba2d_102e_a674_a3bf_fdf79d07223c_MD0008_00 Knockout organisms have one or more genes that have been made inactive. Genes may be knocked out by introducing mutations that result in a truncated gene product, a frame-shifted gene product, or no gene product, each of which results in genes that produce nonfunctional proteins. The biological function of a gene can be inferred by knocking it out and observing the differences between wild-type organisms and knockout organisms. If cldnk is required for wild-type myelination of oligodendrocytes, then knocking the gene out should result in impaired or abnormal myelination. Therefore, comparing myelination of oligodendrocytes in wild-type zebrafish to myelination in cldnk knockout zebrafish would show whether cldnk is required for proper myelin formation. (Choice A) If cldnk is involved in myelination, it is possible that overexpression of cldnk would cause increased myelination. However, it is also possible that endogenous cldnk already yields maximum activity, in which case excess cldnk would have no effect. Therefore, overexpression of cldnk would not necessarily give any information about its role in myelination. (Choice B) Although cldnk expression in cells other than oligodendrocytes could cause myelin to form in those cells, it is unlikely because other gene products are most likely required for myelination as well. (Choice D) A form of the cldnk gene that inhibits activity of the wild-type gene (ie, a dominant negative gene) could show whether cldnk is required for myelination if it were expressed in oligodendrocytes, but not in cells other than oligodendrocytes. Educational objective: A gene's biological function can be inferred by comparing the differences in organisms with the gene knocked out (inactivated) to wild-type organisms.