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Show 154 EFFECTS OF THE CULTURING PROCESS ON THE GENE EXPRESSION OF CULTURED DRGS John Bagley, Kyoungsun Rha (E.C. Bang, Ron Hughen, Alan R. Light) Department of Anesthesiology University of Utah research posters on the hill spring 2012 Effects of the Culturing Process on the Gene Expression of Cultured DRGs John Bagley, Kyoungsun Rha, E.C. Bang, Ron Hughen, and Alan R. Light Department of Anesthesiology Ratio of mRNA expression between stages of the culturing process and a whole DRG, with 1 representing the baseline of comparison. An example of a mouse DRG cell culture used in single cell collection and calcium imaging. Note round neurons which are the cell bodies of sensory neurons. The yellow coloring represents retrograde neuron labeling with Di-I from hind-limb injections. Ratio of mRNA expression between single cell collection (involving primarily muscle sensory neurons) and the 18 hour incubation stage (involving all types of cells present in a DRG), with 1.00E+00 representing the baseline. *These data indicate that genes that detect acid (ASIC genes) and ATP (P2X genes) are greatly enriched in sensory neurons innervating muscle, and thus, may be used to detect muscle pain and fatigue. **Interestingly, the gene for the mu-opioid receptor (OPRM1) is also enriched in sensory neurons innervating muscle, suggesting that opioids may be especially effective for treating muscle pain and may also affect muscle fatigue. John Bagley Kyoungsun Rha Ron Hughen Alan R. Light Antranik.org INTRODUCTION: During the culturing process of mouse dorsal root ganglion (DRG) neurons for research studies on gene expression, many chemicals are used such as trypsin and GDNF, along with a mechanical manipulating process called trituration. Beginning with euthanasia and continuing until the end of the culturing process, the gene expression within DRG neurons may be affected in some unknown way. It is hypothesized that gene expression of mouse DRG neurons is negatively affected during the culturing process due to chemicals such as trypsin and GDNF, trituration, the amount of time passed from euthanasia to single cell collection. These changes are seemingly unavoidable due to the inability to remove and dissociate neurons from whole DRGs of living mice. Identifying the main source of affect may minimize the negative effects created by the culturing process, thus allowing more accurate research data. METHODOLOGY: • Remove eight lumbar-region DRGs from 2-3 week old mouse • Collect one whole DRG in 200uL buffer RLT solution and quickly freeze • Cut DRGs into 2-4 pieces and perform the trypsin reaction, followed by trituration at a volume of 1700uL • Collect 200uL of cells in 500uL buffer RLT and quickly freeze • Pre-plate the remaining cell suspension for one hour • Collect 200uL of cells in 500uL buffer RLT and quickly freeze • Flood the remaining cells in GDNF and incubate for 12-18 hours • Following the incubation, collect the cell suspension in 500uL buffer RLT and quickly freeze In comparing the RNA data of each of these four collected samples, it will be possible to determine which steps in the culturing process most strongly affect changes in gene expression. Modifications to the culturing protocol can then be made to aid in obtaining more accurate results for further DRG studies. Diagram showing the spinal nerve connections, including the dorsal root ganglion (DRG). CONCLUSIONS: 1) The time involved during the culturing process from euthanasia to single cell collection does not necessarily have a negative effect on gene expression. Many of the genes selected for were actually largely enriched in single cell collection (the final stage of the culturing process), suggesting that extended time can positively influence transcription and translation. I. ADRA2A, ASIC1, ASIC3, P2X3, P2X4, and P2X5 were all enriched at least three-fold by the final stage of the culturing process. II. By the end, ASIC2, CGRP, OPRD1, OPRM1, and TRPC1 were all somewhat enriched from the original, whole DRG. III. Only ADRA2C, TRPA1, TRPM8, and TRPV1 were expressed less in single cell collection than in the original DRG. 2) Individual genes each react differently to various stimuli during the stages of the culturing process, thus preventing a generalized affect to be determined for all genes. Therefore, each gene must be taken into account individually when considering changes to the culturing protocol. 3) Some genes change from stage to stage during the culturing process, while some remain unaffected by certain stimuli. I. CGRP remained nearly unchanged through the stages of the culturing process until single cell collection, suggesting that this gene is enriched in muscle neurons only. II. Many non-neuronal cells flourished during the culturing process, which resulted in high enrichment levels of particular genes found more commonly in non-neuronal cells. 4) The culturing process used with mouse DRG neurons does not completely destroy any genes, nor does it introduce any unexpected genes (at least not within the range of genes selected for in this case). During the culturing process of mouse DRG neuron cells for research studies on gene expres sion, many chemical and mechanical processes are used. Beginning with euthanasia and continu ing until the end of the culturing process, cells are affected in some unknown way. It is unclear the effects various chemicals have on gene expression during this process. It is hypothesized that gene expression of mouse DRG neuron cells is negatively affected during the culturing process due to the trypsin reaction, the mechanical manipulation present during trituration, the amount of time required from start to finish, and the presence of GDNF. These changes are seemingly unavoidable due to the inability to remove DRG neurons from live mice, yet it is predicted that the negative effects can be minimized by identifying the main source of affect. A protocol was developed by Ron Hughen and E.C. Bang to discover which step of the culturing process af-fects gene expression most strongly. Eight lumbar-region DRGs are removed from a 2-3 week old mouse. One whole DRG is quickly frozen to preserve the original gene expression. The remaining DRGs undergo a trypsin reaction and trituration, at which point 200uL are removed and quickly frozen to prevent further changes in gene expression. The remaining cell suspension is pre-plated for one hour and another 200uL sample is collected and frozen. Finally, the remaining cells are plated, flooded in GDNF, and incubated for 12-18 hours. Following incubation, the remaining cells are collected and quickly frozen for preserva-tion until RNA extraction occurs. In comparing the RNA of each of these four samples, it will be possible to determine which steps in the culturing process affect changes in gene expression most strongly. Changes can then be made to the culturing protocol to aid in obtaining more accurate results for further DRG studies. |
