Abstract:

Neuropathic pain is chronically increased pain and heat sensitivity resulting from damage to the central or peripheral nervous system. Neuropathic pain is constant, and difficult to treat, because the pain persists even after the injury has healed. Current drug treatments are ineffective, have short durations, and have unpleasant side effects. The SIP-30 gene is a candidate gene for involvement in neuropathic pain. When neuropathic pain is present, SIP-30 levels rise indicating some cause and effect relationship. Transposons are mobile genetic elements that move around within a genome and integrate randomly, in a mechanism similar to some viruses. The PiggyBac transposon, derived from the cabbage looper moth Trichoplusa ni, is a particularly useful transposon, with many features that make it an ideal vector for gene therapy research. Plasmid vectors will be constructed, one carrying the SIP-30 gene, and another carrying antisense SIP-30 in the PiggyBac vector, in order to deliver the genes into mouse nerve cells. This is hypothesized to increase and decrease the expression of neuropathic pain respectively. The gene will be delivered through intrathecal injections into the mouse intrathecal fluid of the spinal cord. If successful, this research can have serious implications for future human gene therapy to treat neurological disorders, using the PiggyBac vector as the gene delivery system.

Introduction:
Features of the PiggyBac Transposon: Transposons are mobile genetic elements that move around within a genome and integrate randomly, in a mechanism similar to some viruses. The PiggyBac transposon, derived from the cabbage looper moth Trichoplusa ni, is a particularly useful transposon, with many features that make it an ideal vector for gene therapy research. The PiggyBac transposon can carry very large sequences; sequences can be as long as 9.1 kb without any noticeable change in efficiency, and sequences as large as 14.3kb have been observed to transpose in mammalian cells. Because the PiggyBac transposon integrates randomly, it integrates into introns and other non-coding sequences 97% of the time, and therefore does not usually disrupt functional genes. As a result, PiggyBac has widespread integration throughout the genome. Furthermore, PiggyBac does not produce the excision footprint mutation that is common to many transposons. Collectively, these features make PiggyBac an ideal vector system for gene therapy research, especially in terminally differentiated cells, such as neurons in the Central Nervous System.

Application to Neuropathic Pain: In my particular project, I will use the PiggyBac vector to test its possible use in the gene therapy of neuropathic pain. Neuropathic pain is chronically increased pain and heat sensitivity resulting from damage to the central or peripheral nervous system. My study will focus on sciatic nerve damage-induced neuropathic pain, which results in gene expression malfunction in the spinal cord.
Neuropathic pain is constant, and difficult to treat, because the pain persists even after the injury has healed. Current drug treatments are ineffective, have short durations, and have unpleasant side effects. The SIP-30 gene is a candidate gene for involvement in neuropathic pain. When neuropathic pain is present, SIP-30 levels rise indicating some cause and effect relationship. SIP-30 is hypothesized to function in the regulation of vesicle exocytosis, though its cellular function remains unknown. I will use the SIP-30 gene in this study in order to target neuropathic pain in a mouse model. This project focuses on the treatment of neuropathic pain, which results from peripheral nerve-induced molecular malfunction in the spinal cord. Since the cells involved in the spinal cord are part of the central nervous system, they do not replicate. This makes gene therapy treatments more difficult, since the cells do not replicate and amplify the genes that they are treated with. The PiggyBac transposon integrates into the host cell genome, therefore allowing itself and any gene it carries to be expressed at moderate but stable levels. This makes it possible for SIP-30 to be expressed to test its role in mediating neuropathic pain. It is our hypothesis that this will allow PiggyBac to be a particularly effective vector for gene expression in nerve cells.

Materials and Methods:
Plasmid Vector The plasmid vector, which will be used in the Intrathecal injections, will consist of the PiggyBac backbone and the beta-galactosidase gene. The PiggyBac backbone is 5.2kb long, and has the BamHI and HindIII restriction sites, which will be important when it comes time to ligate the backbone to the lac Z gene. In addition, the PiggyBac backbone has a sequence for the Act-RFP gene which codes for the red florescent protein marker. This gene will be removed and replaced by the lac Z gene, which will be used as the reporter gene instead. The lac Z gene comes from the beta-galactosidase vector. This vector is 4.1kb long and contains the EcoRI and BamHI restriction sites around the lac Z gene. Subsequently, the SIP 30 gene and RNAi will replace the lac Z gene.

Transformation of cells In order to amplify the PiggyBac plasmids, the DNA must be transformed into bacteria, which will replicate the plasmid DNA through their own replication. The cells used in the transformation were NEB B10Beta E. coli cells. These cells were thawed on ice for 10 minutes. The process is done gradually, in order to protect the transformation efficiency. The undiluted PiggyBac plasmid was added to 50 µl of the cells at volumes ranging from 2 µl to 10µl. The cells were then incubated on ice for 30 minutes to absorb the DNA. The cells were heat shocked for 30 seconds at 42 degrees Celsius, and then allowed to recover on ice for 1-2 minutes. 800 µl of SOC medium was added to these cells, followed by a 37 degree Celsius water bath for 1 minute. These cells were then incubated at 37 degrees Celsius with gentle shaking for 45 minutes. When these cells were done incubating, 200 µl of them were plated on ampicillin and SOB medium Petri dishes and placed in the 37 degree Celsius incubator overnight. The following morning, the plates were taken out of the incubator, the colonies were counted, and the plates were stored in the 4 degree Celsius freezer for future use.

Plasmid Minipreparation by the Alkaline Lysis Method Once colonies for all the PiggyBac plasmids were obtained, the next step was to isolate the plasmid DNA from the bacterial cells. This is done through a miniprep. The first method I used for the miniprep was the Alkaline Lysis method. The first step is to pick a colony from the plates, and grow it in Lb medium with ampicillin, and incubate it overnight at 37 degrees Celsius with vigorous shaking. The following morning, 1000 µl of these cultures were placed in an Eppendorf tube, which was placed in the microcentrifuge and spun for 1 minute at the maximum speed, 15,000 rpm. Afterwards, the supernatant was discarded, and 250 µl of the Qiagen Minikit P1 buffer was added to the cell pellet. This buffer resuspends the cells. These cells are then lysed by adding 250 µl of Buffer P2. When the cells are lysed, the solution turns to a dark blue color. The solution is then neutralized using 350 µl of Buffer N3. This causes a white precipitate to form. These cells are then microfuged for 5 minutes at maximum speed. This forms a pellet of cell debris, and a supernatant containing the DNA. This DNA can late be purified by several methods.

Plasmid Minipreparation by Boiling Method Alternatively, the overnight culture containing the PiggyBac plasmids can be isolated through the boiling method. The first step is to resuspend the pellet in 100 µl of STELT buffer (95 % STET Buffer and 5% Lysozyme). These cells are then boiled for 60 seconds in a water bath. Following this, the cells are microfuged at maximum speed for 10 minutes. The supernatant is then transferred to 80 µl isopropanol, followed by microfuging it again for 10 minutes. The pellet can then be used for DNA extraction.

DNA extraction using Qiagen minikit Spin Columns In order to extract the DNA from the solution, the DNA supernatant were added to the spin columns. The spin columns were then centrifuged for 1 minute at maximum speed, and the flow-through was discarded. Afterwards, 750 µl of the PE Buffer was added to the spin columns, which were centrifuged again for 1 minute at maximum speed. The flow-through was drained, and then the spin column was spun again at maximum speed for 1 minute to remove any residual wash. Following this, 60 µl of EB buffer was added to the column to elute the DNA. The solution was incubated at room temperature for 1 minute before it was centrifuged at maximum speed for 1 minute. The flow through is the final DNA solution, which was stored in 4 degrees Celsius for future use.

Ethanol Precipitation of DNA The final supernatant from the Alkaline Lysis miniprep can be treated chemically to extract the DNA. 400 µl of the supernatant was mixed with 1/10 its volume of NaAC (3M) pH 5.2. Then, twice the volume of 95% ethanol is added, and the solution was mixed. The solution was chilled in -20 degrees Celsius for 45 minutes. Subsequently, the solution was microfuged at maximum speed for 15 minutes at 4 degrees Celsius. The liquid was then removed, and replaced by an equal volume of 70% ethanol. The ethanol was then removed by pipette, and the pellet was left to dry for 2 hours. Finally, the pellet was dissolved in 20-50 µl of TE buffer with RNase A.

Phenol Chloroform and Ethanol Precipitation Another method of DNA Precipation is using Phenol Chloroform and Ethanol. First, the Phenol Chloroform bottle was shook, then 1.5 ml was transferred to each tube. This tube was centrifuged for 1 minute at maximum speed and room temperature. The bottom layer of the centrifuged solution was transferred to a fresh tube. The aqueous and organic phases were vortexed, then centrifuged for 2 minutes at 4 degrees Celsius at maximum speed. An equal volume of the upper aqueous layer was pippeted and transferred to each plasmid sample. Twice the volume of 100% ethanol was added to the solution, which was then incubated for 5 minutes at room temperature. The samples were then centrifuged for 5 minutes at 4 degrees Celsius and maximum speed. The supernatant was carefully removed by pippetting it twice. 1ml 70% Ethanol was added to each sample. The samples were again centrifuged, this time for 2 minutes at maximum speed and 4 degrees Celsius. The supernatant was removed, and the DNA pellet was mixed with 50µl TE + RNase A in each sample. The samples were stored and frozen in -20 degrees Celsius.

Restriction Digestion A restriction digestion was performed on the purified DNA to test if the correct size plasmids were present, meaning the correct DNA was isolated without too much contamination. First, a digestion mix was made. The digestion mix is made up of 15 µl Water, 2 µl 10 X Enzyme buffer, 2 µl plasmid DNA, and finally 1 µl total volume of the appropriate enzyme, or .5 µl of two appropriate enzymes if a double digestion was necessary. Each Plasmid requires a different enzyme and buffer. PB[Act-RFP]DS requires HindIII, BamHI, and NEB buffer 2. PB[SV40-neo] requires AscI and NEB buffer 4. PB[PGK-neo] requires EcoRI and NEB EcoRI buffer. Act-PBase requires HindIII, XbaI and NEB buffer 2. CMV-PBase requires HindIII, EcoRI, and NEB buffer 2. After the digestion mixes were made, they were mixed, then incubated for 1 hour at 37 degrees Celsius. Another mix was made for all the plasmids without the enzymes, called the uncut mix. This was used as a means of comparison when the gel electrophoresis was performed.

Agarose Gel Electrophoresis The first step in performing agarose gel electrophoresis is to prepare the gel. The gel was made by mixing 130 ml TAE buffer with 1.3g agarose. This solution was microwaved for 1-2 minutes until the agarose was fully dissolved. 9 µl Ethidium Bromide was added to allow the DNA to fluoresce under Ultra Violet light. Next, the gel was poured into a gel box, and a comb was inserted to form the wells. The gel was washed in TAE buffer. The gel was left to sit for 20-30 minutes for the gel to solidify and the wells to form. 2 µl loading dye was added to each DNA sample. The samples were loaded into the wells, along with the DNA ladder. 100 volts was applied to the gel for 45 minutes, and the results were recorded.

Objectives:
Construction of Positive Control Vector: The first step of this study is to construct the vector that will deliver the gene of interest. Before the SIP-30 is used, a test vector must be constructed to determine if PiggyBac can in fact transpose effectively into cells of the central nervous system as hypothesized. The pSV-(-Galactosidase plasmid contains the Lac Z gene, which dyes blue in the presence of the X-gal substrate and serves as a very sensitive marker for the expression of the transfected gene. The Lac Z gene will be isolated from the pSV-(-Galactosidase plasmid and inserted into the PiggyBac backbone in order to make the complete test vector. Plasmid DNA will be isolated by preparation from transformed E. coli cells. The isolated DNA will be subjected to restriction endonuclease digestion and the digested DNA will be separated by agarose gel electrophoresis. The desired band of DNA, which carries the Lac Z gene, will be isolated from the gel and will be used for insertion into the PiggyBac backbone vector. Parallel to the Lac Z gene isolation, the PiggyBac vector DNA will also be prepared and linearized with the appropriate restriction endonucleases. These two pieces of DNA, the DNA insert and the PiggyBac backbone, will be joined together using DNA ligase to make the final test vector.

Results:
Preparation of Competent Cells: In order to perform the transformation of cells, it is necessary that the cells to be transformed are made competent. This was done with treatment with the Inoue Buffer. This buffer consists of 800 ml Water, 10.88 g MnCl2 tetrahydride, 2.20 g CaCl2 dihydride, 18.65 g KCl, and 20 ml PIPES solution. The PIPES solution was prepared by dissolving 15.1 g PIPES in 80 ml Water, then adjusting the pH with 5M KOH, then adding 20 ml Water. Water was added to the Inoue Buffer until the total volume was 1 liter. This buffer was sterilized through a .45 µm Nalgene Filter. The filtered solution was divided and stored at -20 degrees Celsius for future use. The cells that were going to be made competent were plated at 37 degrees Celsius for 16-20 hours. The following day, I picked a colony from the plate and transferred it to 25 ml LB medium. This was incubated at 37 degrees Celsius for 6-8 hours with vigorous shaking. This was then divided into three flasks: the first was filled with 10ml, the second was filled with 4ml, and flask 3 was filled with 2ml. These flasks were incubated overnight at room temperature with moderate shaking. The OD600 was measured to determine when the growth was sufficient. Once the OD600 reached 0.55, the culture was transferred to an ice water bath for 10 minutes. The cells were then centrifuged at 3900 rpm at 4 degrees Celsius for 10 minutes. The media supernatant was discarded, and the cells were resuspended in 20 ml of the Inoue Buffer. 1.5 ml DMSO was added, and then the solution was stored in ice for 10 minutes. The solution was then divided into aliquots of 50-100 µl. these aliquots were snap frozen in liquid nitrogen, and then stored in -80 degrees Celsius until they were ready for use. A test transformation was performed to calculate the transformation efficiency of the competent cells. This was performed by transforming the DNA at concentrations varying ten-fold from .1 pg/µl to .1 ng/µl. In addition, commercial B10-Beta commercial competent cells were used as a positive control, and cells with no DNA were used as a negative control. By counting the colonies formed from the transformation, the transformation efficiency can be calculated. The DNA used for the transformation was the plasmid PSV/CMV. The colonies were counted and the results were as follows:

|Plate / Number of Colonies |Transformation Efficiency |
|Commercial.1 pg/µl – 6 colonies |3 x 107 cfu/µg |
|1 pg/µl – 6 colonies |3 x 106 cfu/µg |
|.01 ng/µl – 16 colonies |8 x 105 cfu/µg |
|.1 ng/µl – 84 colonies |4.2 x 105 cfu/µg |

Results of Transformation 7/30/10: The average of the transformation efficiencies was calculated as 5.6 x 106 cfu/µg.

Transformation efficiency was calculated by dividing the number of colonies by the amount of DNA used for the transformation. By taking the average of all the transformation efficiencies, the transformation efficiency was calculated as 5.6 x 106 colony forming units per microgram of DNA.

Transformation of PiggyBac Plasmids: Once the transformation efficiency was known, the competent cells were used to transform the PiggyBac vectors for amplification. At first, since the concentration of the PiggyBac vectors was unknown, they were serially diluted to concentrations of 1:10 and 1:100 before the transformation. However, when the transformation was performed, none of the plates showed any growth, implying that the concentration was very low. The DNA concentrations of the undiluted samples were about .1 pg/µl. The transformations were redone using 4 µl DNA, resulting in colonies 1 colony on one plate, and 2 colonies on another plate. The transformation was then redone using 6 µl DNA, resulting in 1 colony on one plate, 4 colonies on another plate, and 57 colonies on a third plate, while the rest still had no growth. For the plasmids that still produced no colonies, the transformation was redone while plating 400 µl of the culture instead of 200 µl. This resulted in a few colonies on each plate, and as a result, all the plates had a few colonies and were ready for plasmid minipreparation.

Plasmid Minipreparation of PiggyBac: Once colonies were isolated for all of the PiggyBac plasmids, it was time to isolate the DNA for amplification. The first method used was the alkaline lysis in compound with spin column extraction. Following this, the nanodrop was used to test the concentration of the plasmids, which ranged from 13.3 ng/µl to 27.6 ng/µl. this yield was not nearly sufficient to perform a restriction digest, so the miniprep was redone. The nanodrop showed that the miniprep yielded similar results the second time. In order to achieve a better yield, I attempted the boiling lysis method for the miniprep. Unfortunately, this method yielded no pellet after the isopropanol was added. The alkaline lysis was then reattempted, however, ethanol precipitation was used instead of the spin columns this time. When the concentration was measured using the nanodrop, at first glance it appeared to have a very strong yield. The concentrations ranged from 135.5 ng/µl to 708.1 ng/µl .
Restriction Digestion and Gel Electrophoresis: The next step was to perform a restriction digestion to determine if the correct size bands would show up in the gel electrophoresis. The gel electrophoresis was a success, since the positive control, the DNA ladders, ran correctly. However, there were no bands in any of the lanes with the PiggyBac plasmids.

[pic]
Results of Gel Electrophoresis 11/18/10: The bands in the marker lanes show that the gel was successful. However, the lack of bands in the Cut and Uncut plasmid lanes suggest a lack of sufficient DNA.

Curiously, there was some minor fluorescence in the wells of a few of the PiggyBac lanes. These results implied that there might not be enough DNA in the PiggyBac samples, so I went back to the nanodrop to see if it could provide any insight. The graph of the UV absorption from the nanodrop shows only a slight hump in the 260-280nm range, when there should be a large peak if there is a large quantity of DNA.

[pic]
Results of Nanodrop 12/10/10: The lack of a peak at around 260 nm suggest that the UV absorption was due to contaminants, and not the desired plasmid DNA.

Plasmid Minipreparation by Alkaline Lysis Method For improved results, the minipreparation was performed using freshly made reagents. This was done with the intention to maximize the yields. First, the overnight cultures containing the desired plasmids were centrifuged to remove the broth supernatant. The cell pellet of each sample was resuspended in 100µl of resuspension Buffer I. Buffer I was prepared with 50mM Glucose, 25mM Tris-Cl (pH 8.0), and 10mM EDTA (pH 8.0). The combined solution was autoclaved for 15 minutes at 15psi. Next, 200µl of Alkaline lysis Buffer II was added to each sample. Buffer II was prepared with 20µl 10M NaOH, 100µl 10% SDS and 880µl H2O. The final concentration was .2M NaOH and 1% SDS. Once Buffer II was added, the samples were gently inverted to mix the solution without damaging the DNA. Next, 150µl Neutralization Buffer III was added to neutralize the samples. Buffer III contains 60ml 5M Potassium Acetate, 11.5ml Glacial Acetic Acid, and 28.5 ml H2O. The tubes were gently inverted to mix, and then incubate on ice for 5 minutes.
Second Plasmid Preparation: Once it was determined that there was not sufficient DNA in the samples, the plasmid minipreparation was attempted again to achieve a better yield. This time, a new method was selected to improve the results. We performed the Alkaline Lysis, this time using fresh reagents. In addition, a new method of DNA precipitation was needed, since the previous methods failed to give sufficient yields. This time, we used the phenol chloroform and ethanol precipitation. The results this time were much better than previously achieved. The DNA concentrations improved by over ten-fold for most of the plasmids. The concentrations as measured by the nanodrop were the following:
| |260/280 |Concentration |
|PB1 |2.04 |1.24µg/µl |
|PB2 |2.04 |1.64µg/µl |
|PB3 |1.91 |0.71µg/µl |
|PB4 |2.02 |1.46µg/µl |
|PB5 |1.95 |0.39µg/µl |

Results of Nanodrop 2/8/11: These nanodrop results suggest a large amount of DNA present. The DNA appears to be sufficient for a gel electrophoresis.

Second Restriction Digestion and Gel Electophoresis: First, all the DNA samples were diluted to .4µg/µl. These were then mixed with the proper buffers and Restriction Endonucleases. In addition, uncut control samples were prepared with DNA and buffer, but no enzymes. The samples were run in the Gel electrophoresis, the results were as follows:
[pic]
Results of Gel Electrophoresis 2/25/11: The bands in the marker lane show the gel was again successful. However, the lack of bands in any other lane suggest that once again there is not enough plasmid. DNA is present in the samples.

DNA appeared in the ladder lane, but not in any of the sample cut or uncut lanes. The restriction digest and gel electrophoresis were attempted again, this time using diluted enzymes, and undiluted DNA. The results were as follows:

[pic]
Results of Gel Electrophoresis 3/4/11: The bands in the marker lane show the gel was again successful. However, the lack of bands in any other lane suggest that once again there is not enough plasmid. DNA is present in the samples.

Once again, there was DNA in the Ladder lanes, but not in any of the sample lanes. This implied that there was not enough DNA in the samples. To test if the DNA was lost, the remains of the sample were tested using the nanodrop. The results were approximately the same, indicating the DNA loss was not the issue.

[pic]
Results of Nanodrop 3/22/11: There is a strong peak at 260 nm, suggesting that there is a lot of DNA in the sample. However, the gel electrophoresis contradicts these results.

Discussion: The gel electrophoresis was successful once again, however, the correct bands of our desired DNA have yet to be isolated. The nanodrop indicates that the concentration of DNA is sufficient, yet the results of the gel electrophoresis contradict this, indicating that there is not sufficient DNA in our samples. One possible explanation is that the DNA in our samples is a contaminated mixture of nucleic acids, which would give good results in the UV fluorescence test of the nanodrop, but poor results in a gel electrophoresis. Furthermore, all the DNA in the samples was used up, so new DNA must be isolated, and the minipreparations must be redone. Once the DNA is isolated from the miniprepations, it must again be tested for the proper fragments using restriction digestion and gel electrophoresis. Once the correct bands are successfully isolated, I can then proceed to perform a maxipreparation of the plasmids, to isolate a larger amount of the plasmids. This will then be used to construct the complete plasmid vector with the PiggyBac backbone, lac Z gene, and the oligonucleotide linkers. Once this vector construction is complete, it will be tested in mice through Intrathecal injections, spinal cord extraction, and x-gal staining. After it is shown that the PiggyBac transposon can successfully transpose itself into mice nervous system cells, the lac Z gene will be replaced with SIP 30 and RNAi, to test if this transposon can be used to treat neuropathic pain in mice. SIP 30 will overexpress neuropathic pain, while RNAi should block the expression of neuropathic pain. After the PiggyBac vector shows success in treating neuropathic pain when intrathecally injected into mice spinal cords, it will be tested in mice brains.

Vector Construction and Future Implications: When the test vector construction is complete, it will be administered to mouse spinal cord by intrathecal injection. After allowing various time for gene expression, the spinal cord will be dissected. Successful PiggyBac transposition and subsequent Lac Z gene expression will be tested with X-gal staining; if the transposition is successful, the spinal cord should turn blue. Once PiggyBac is shown to successfully transpose into cells of the central nervous system, two new vectors will be designed to alter the expression of neuropathic pain gene in mice. The first vector will consist of the PiggyBac backbone with the SIP-30 gene as the insert. When this is given to mice, it should result in increased neuropathic pain. The second vector will consist of the PiggyBac backbone and antisense SIP-30 as the insert. This should inhibit the expression of SIP-30 gene, and therefore reduce neuropathic pain. This research has many important implications and applications to medicine. If transposition occurs successfully in the spinal cord and alters the expression of neuropathic pain, not only will this be important to gene therapy for neuropathic pain, but more generally, it will also present a new method of stable, long-term alteration of gene expression in cells of the central nervous system, which is currently very difficult. This can be used to treat a wide variety of neurological disorders that are currently difficult to treat.

References: Ding, S., Wu, X. Li, G., Han, M., Zhaung, Y., and Xu, T. 2005. Efficient Transposition of the piggyBac (PB) Transposon in Mammalian Cells and Mice. Cell 122: 473-483.

Yu-Qiu Zhang, Ning Guo Guangdun Peng, Mei Han, Jeremy Raincrow, Chi- hua Chiu, Lique M. Coolen, Robert J. Wenthold, Zhi-Qi Zhao, Naihe Jing, and Lei Yu. "Role of SIP30 in the Development and Maintenance of Peripheral Nerve Injury-Induced Neuropathic Pain." Pain 146.1-2 (2009): 130-140.