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Volume 1 November 2012

MURJ Mission Undergraduate Research Journal

STEM-MURJ Volume 1, June 2012

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Los Angeles Mission College Title III, STEM (Science, Technology, Engineering and Math) Program

Mission Undergraduate Research Journal

MURJ

Volume 1 November 2012

Publisher Los Angeles Mission College STEM Program in collaboration with Title V-‐ISSA (Improving Student Success and Access) Program, Science Success Center LAMC Faculty Lead: Michael Reynolds STEM Director: Parvaneh Mohammadian Title V Assistant Dean: Young-‐Ji Lee Editor: Lilit Haroyan ©2012 Los Angeles Mission College, STEM http://lamission.edu/stem/ STEM and Title V Programs are funded by the U.S. Department of Education


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The mission of the Mission Undergraduate Research Journal (MURJ) is to encourage, recognize, and

reward students’ academic activity outside the classroom, while providing an opportunity for the sharing

of research and ideas. MURJ strives to encourage students to become interested in science research by

providing a forum for studied work and knowledge between the STEM disciplines


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Letter From the Editor Dear Reader,

It is with great pride and anticipation that we publish the first volume of Mission Undergraduate Research Journal (MURJ) at Los Angeles Mission College (LAMC). LAMC STEM Program is very pleased to present undergraduate science research done by LAMC students.

Enclosed in this journal are articles written by members of summer 2012 internship program. The articles represent the results of work performed over a twelve-‐week period. All students participating in the program experienced scientific writing for the very first time.

The STEM Program plans to expand and formalize the undergraduate research activities at Mission. It is determined to expand the undergraduate research in other STEM disciplines and plans to contribute to the Physical and Life Sciences.

LAMC STEM faculty and program team are dedicated to the effort. We would like to especially thank Professor Michael Reynolds, Life Sciences Department Chair, whose guidance and dedication were crucial in piloting the summer 2012 internship program. We also want to thank Professors Aida Metzenberg, Stan Metzenberg, Randy Cohen, and Ray Hong from California State University, Northridge for opening the doors and providing meaningful learning experiences for our students.

It is our hope that MURJ will serve as an inspirational tool to our students and faculty. The undergraduate research opportunities will nurture a community of students in their pursuit of excellence.

Sincerely, Lilit Haroyan, Editor November, 2012


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Contents

A Letter from the Editor 3

Non-‐Viral Vectors and the Trojan Horse as Therapeutic Agents for Mucopolysaccharidosis type 2 John Daniel David

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Exercise and Its Effects on the Spastic Han-‐Wistar Rat with Ataxia Natalie Derkrikorian 10

The Extraction and Identification of Wild Nematodes Through the Use of the Small Subunit 18 (SSU 18) gene Veronica Guizar

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Gene Therapy Using A Non-‐Viral Vector For Mucopolysaccharidosis Type 2 Eduardo Martin 21


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Non-‐Viral Vectors and the Trojan Horse as Therapeutic Agents for Mucopolysaccharidosis

type 2 John Daniel David, Sponsored by Professors Aida Metzenberg and Stan Metzenberg, Department of Biology, California State University, Northridge, 18111 Nordhoff Street, Northridge, California 91330

INTRODUCTION Lysosomes can be best described as the waste center of a cell. They break down waste material and cell debris. Lysosomes contain enzymes which do the work of breaking down the waste located in the cell. Lysosomal Storage Disorders, also known as LSD’s, are primarily the result of functionally defective enzymes within the lysosomes. Mucopolysaccharidosis is a family of LSD’s, in which there are 9 metabolic disorders that comprise the various types of mucopolysaccharidosis (MPS). These nine metabolic disorders are due to certain enzyme inactivity in the lysosomes that prevent them from breaking down important glycosaminoglycans. Glycosaminoglycans are long chains of sugar carbohydrates that are responsible for things such as helping build bones, cartilage, tendons, corneas, skin and connective tissue. Mucopolysaccharidosis Type 2 (MPS2) is commonly known as Hunter’s Syndrome and it arises from insufficient or lack of Iduronate 2 Sulfatase (I2S) due to mutations within the gene that codes for it. Since MPS2 is caused by a lack of I2S, this experiment will also consist of injecting the I2S enzyme into the patients. A method called the Trojan Horse will also be used in order to deliver the I2S across the blood-‐brain barrier. The non-‐viral vectors that are going to be studied include pEPito and pUMVC3 which will be used to help promote the IDS gene that will be placed into the MPS2 patients.

MATERIALS AND METHODS Competent cells from XL Blue E.Coli cells 1. Inoculate a single E. Coli colony into 5 ml of LB Broth from an agar plate labeled XL Blue. 2. Shake at 37 ºC at 50 rpm overnight. 3. The next day, take 50 ml of LB Broth and pour into a 500 ml Erlenmeyer flask. 4. Add 1ml of your overnight growth to the flask containing 50ml LB Broth.


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5. Take spectrophotometric reading at time zero at wavelength 600 nm. 6. Place flask in shaking incubator at 37 ºC and 50 rpm, making sure to not completely seal the flask with a cap to allow for aeration. 7. Take spectrophotometric readings hourly at 600 nm until the absorbency reading is close to 0.6 at which point the readings should be taken every 5 minutes (600 ul aliquots) 8. Once the absorbency has reached 0.6 (or significantly close), divide the cells into two 50 ml falcon tubes (about 5 ml into each). 9. Centrifuge at 2000 xg for 5 minutes. 10. Discard the supernatant and add 5 ml of ice cold 0.1M CaCl to the pellet (0.5 ml of CaCl into each falcon tube). 10. Gently dissolve the pellet and transfer all the contents into one of the two falcon tubes. 11. Centrifuge at 4000xg for 5 minutes. 3. Discard the supernatant and add 5ml of cold 0.1M CaCl. 4. Add glycerol to 5% (meaning add 80 µl of glycerol to falcon tube containing ml of CaCl and pellet) 5. Divide competent cells into 0.1 ml aliquots in 0.5 ml tubes (glass tubes preferred) 6. Store at -‐70 ºC. Transformation of competent cells in order to clone pEPito. 1. Label two 15 ml tubes as “pEPito” and as “pUC19”. Place both on ice. 2. Turn on the water bath to 42 ºC. Proceed to Step 3 only when at 42 ºC. 3. Thaw out 200 µl of competent cells and gently mix. 4. Place 100 µl of competent cells into each falcon tube. 5. Add 1 µl of pEPito (50pg/µl) to the first tube. Make sure to move the pipette through the cells while dispensing the contents in a circular motion. Gently tap the falcon tube to mix. 6. Repeat step 3 with the control vector pUC19. 7. Place both tubes in ice for 30 minutes. 8. Place your medium (SOC) at room temperature. 9. Heat shock cells by placing tubes in a 42 ºC water bath for 45 seconds. (Note: Be very careful to not bump the tubes.) 10. Place tubes in ice for 2 minutes. 11. Add 900 µl of SOC medium to each of the falcon tubes. 12. Shake tubes at 37 ºC at 225 rpm for 1 hour. 13. Obtain 6 plates of LB Agar and label the plates: pEP A, pEP B, pEP C, pUC19 A, pUC19 B, pUC19 C 14. Add the following to a sterilize microtube: 25 µl of IPTG, 25 µl of X-‐Gal, 25 µl of Ampicillin. 15. Plating: Add your 75 µl solution from step 14 onto LB Agar Plate; Dip the glass spreader in 70% ethanol; Flame the rod; Allow for the rod to cool for about 10-‐15 seconds; Spread the contents throughout the plates. 16. Let the plates dry for 15 minutes. 17. Add 1 µl of Transformed cells to plate A 18. Add 10 µl of Transformed cells to plate B 19. Add the remainder of your Transformed cells to plate C 20. Repeat steps 16 – 18 for pUC19 21. After 10 minutes, flip all plates labeled A and B. Allow plate C to dry longer before flipping. 22. Incubate overnight at 37 degrees ºC.


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23. Next day, if white colonies present, take one single colony and place in 5mL of LB at 37C at 240rpm for 12hrs. 24. Also, take one colony and inoculate a slant by stabbing. 25. Perform Plasmid purification aka Wizard PCR For Ligation-‐Independent Subcloning Using 40mer Primers. 20 of which match the vector and 20 of which match the insert Standard Reactants Standard Reactants Volume (µl) di H20 36.0 5x Phusion HF Buffer (7.5 mM Mg +2) 10.0 dNTPs (25mM) 0.5 Primer 1 (5pmol/ µl) 1.0 Primer 2 (5pmol/ µl) 1.0 Phusion DNA Polymerase (2U/ µl) 1.0 PCR Conditions: 50uL reactions, 2 holds Temperature Time # of Cycles 97.0 C 5 Seconds No cycles 97.0 C 15 Seconds 10 cycles 55.0 C 15 Seconds 10 cycles 72.0 C 90 Seconds 10 cycles 97.0 C 15 Seconds 25 cycles 72.0 C 90 Seconds 25 cycles 72.0C 5 minutes No cycles 4.0C Infinity No cycles *Clean your area, discard pipette tips appropriately and put everything away. Agarose Gel Electrophoresis http://www.methodbook.net/dna/gelextrc.html 1. Weigh out agarose (a 2% gel weights 2.0grams in 100 ml of 50X TAE Buffer; to be a 1X Final). 2. Microwave to dissolve agarose, being careful not to allow it to boil over. Let it cool until you can touch the flask with your hands. 3. Cover the north and south ends of the gel mold using tape or press and seal. Pour the warm agarose into the gel mold and allow to cool completely until it solidifies. 4. Assemble the electrophoresis apparatus, and connect the electrodes to the power supply. 5. Remove the tape and/or the press and seal from the mold and place the mold on the electrophoresis apparatus. 6. Run the gel at 100 volts for 1 hour. 7. Stain gel in a 1:1,000 dilution of 10mg/ml ethidium bromide stock solution for 10 minutes. Rinse gel in diH20. 8. Irradiate with UV light to reveal the DNA.


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9. Photograph illuminated. Do not discard the gel until the experiment is finished. You may wrap the gel in Saran Wrap and refrigerate to store, but note that over time, the contents of the gel will exit the gel by diffusion. 7.5% PAG Electrophoresis (PAGE) Mini-‐Protean 3 Cell 10.1x7.3cm thin plate (165-‐3308) thick plate (165-‐3310) PAGE – General Calculations Size of gel = Depth x Width x Length For example, a gel might be 10 x 10 cm squared, and 0.75 mm thick. The volume of the gel would be 7.5mL (remember that 1cm = 10mm). Place one of the short glass plates and one of the long glass plates together with a grey rubber spacer between the plates on each side. Place these two glass slides in the green holder. Make sure that the glass slides are evenly aligned in the green holder. Place the green holder along with the glass slides onto the plastic "house." Clamp the plates onto the poring stand. Pore the gel with catalysts (TEMED and APS) between the plates. After polymerization, load the gel. Snap the arms of the green holder together, to ensure a tight seal. 1. Obtain a beaker or flask for each planned gel. The following solutions are to be put in the beaker/ Percentage = Grams / 100 ml 2. Prepare a solution acrylamide:bisacrylamide (20:1 acrylamide:bisacrylamide, molar ratio). 3. For a 7.5% gel, pipette in 4. Bring volume to 5.95ml with nH2O (for a 6.5% on a 7.5ml gel it equals 6.13ml of nH2O) 5. Under the fume hood, for a 7.5 mL gel, add 10 µl of TEMED simultaneously with the addition of 60uL of 10% APS. If the volume is different from a 7.5ml, increase the catalysts or decrease them proportionally. For example, for a 9x10x.75 = 6.75mL gel, 6.5/7.5 X 6.0 = 5.2 µl of TEMED and 31.2 µl of 10% APS (To make the APS solution, by weighing out 0.1 gram of APS and adding 1 mL of nH2O. Make this working solution in a 1.5 mL tube. This can be stored at -‐ 20 degrees for 2 weeks. Please be sure to date the tube). 6. Rapidly pour the gel so that pouring precedes polymerization. 7. Pipette the solution from the beaker or flask between the plates. (Each set up is different.) Be careful not to introduce any air bubbles. Pipette until solution is all the way to the top. Insert the comb(s) (green) immediately into the gel. You will notice solution pouring out, but that’s okay. 8. Let the gel polymerize for about an hour.


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RESULTS

Figure 1. Plates Containing E. Coli

Figure 1 contains 6 plates; three of which contain streaks of bacteria containing the control vector, pUC19, and the three on the bottom contain streaks of bacteria containing the vector we are testing, pEPito. Each plate consist of LB agar and ampicillin. The plates show the amount of solution containing the bacteria that was spread on them. On the bottom row, you can see in red that there is 10 microliters of bacteria solution spread on the plate, 100 on the next, and whatever was left over of the solution on the last plate. The same amounts were spread on each of the corresponding control plates that are shown above the test plates. The plates were incubated overnight at 37 degrees celsius.

Figure 2. Agarose Gel Containing Digested and Undigested pEPito


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Figure 2 displays a 1% agarose gel that was run for 1.5 hours at 90 volts. It contains a ladder the first well. The third well contains undigested pEPito. The fifth well contains pEPito that was digested with two restriction enzymes, SFi1 and Apa1. The seventh well contains pEPito that was digested with a single restriction enzyme, Apa1. The ninth well contains pEPito that was digested with a single restriction enzyme, SFi1. The pEPito that was used in this experiment was purified using Wizard® Plus SV Minipreps DNA Purification System.

DISCUSSION The purpose of the experiment shown in Figure 1 was to transform bacteria so that they would contain the pEPito vector. We know that this worked because the pEPito vector is resistant to ampicillin. As shown, the plates contain ampicillin, but since the bacteria took in the pEPito vector, it was able to grow and multiply. The purpose of the experiment in Figure 2 was to see if we were able to digest pEPito and cut them at certain restriction sites. In our case, the sites were Apa1 and Sfi1. It appears that our experiment did not work. As demonstrated in well 3, there are multiple strands. Since there was no cutting of the DNA in well 3, there should only be one strand of DNA. The other wells also do not seem to have the correct amount of strands. A source of error in this experiment may have either occurred while they were in the PCR machine. The DNA does not seem to have digested properly. Another possibly cause is the run time of the gel using electrophoresis. A longer run time may have made the lines more visible and distinct. Further research can be done to increase the efficiency of cloning bacteria with a desired vector, as we had many problems getting the bacteria to transform correctly. The information obtained in these experiments may lead to finding a better suitable vector for projects pertaining to gene therapy.

REFERENCES Muenzer J. The mucopolysaccharidoses: a heterogeneous group of disorders with variable pediatric presentations. J Pediatr. 2004 May;144(5 Suppl):S27-‐34.

ACKNOWLEDGEMENTS

I would like to thank Dr. Aida Metzenberg for allowing me to work in her lab and to do the various experiments in this paper. I would also like to thank Ozvaldo Larios who mentored me and my fellow interns during the summer. To my fellow interns, Edward Martin, and Joyce Rivera, it was great getting to know you two over the summer and it was great working on these experiments together. Lastly, I would like to thank Professor Mike Reynolds for the opportunity to work in this summer internship at CSUN.


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Exercise and Its Effects on the Spastic Han-‐Wistar Rat with Ataxia

Natalie Derkrikorian, Sponsored by Professor Randy Cohen, Department of Biology, California State University, Northridge, 18111 Nordhoff Street, Northridge, California 91330

INTRODUCTION

Ataxia is a disease that affects millions of people. It presents itself with signs of gate incoordination, tremor, instability, muscle wasting, hind limb stiffness and gaze apraxia. Ataxia results in histopathologic abnormalities, microscopic anatomical changes in diseased tissues, indicating a loss in Purkinje and granular cells. The spastic Han-‐Wistar rat (sHW) suffers from a neurodegenerative disorder, the result of an autosomal, recessive mutation. Due to its mutation, the mutant sHW rat encounters loss of motor coordination, the incapability to walk or feed and an early death at sixty days of age. Studies have shown that exercise leads to an improvement of those suffering from ataxia and cognitive dysfunction. With treadmill exercise, mutant sHW rats show a reduction of degeneration in certain regions of the brain, improving their mobility, balance, and walk. Due to the fact that the sHW rat shows a loss of neurons in the cerebellum, it can be studied to understand the effects of exercise on neurodegeneration in the early stages of ataxias.

MATERIALS AND METHODS The goal of the study was to evaluate the effects of exercise on prolonged existence, physical activity and survivorship of cerebellar Purkinje cells in the sHW rat. Mutant littermate pairs were randomly chosen as non-‐runners and runners in the treadmill exercise study, yet remaining together throughout the duration of the study. Five days a week the runner rats ran on a Columbus Instruments Exer 3Rmotorized treadmill at 15.0m/min on a 15% incline for thirty minutes. Weight gain or loss was monitored every five days over the lifespan of the rats, and prolonged existence was estimated in both the runner and non-‐runner groups. Conditions were slightly altered for runners as they aged and their disease progressed, making it suitable in speed or treadmill slope for the rat to complete its thirty minute run.


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RESULTS Table 1

GROUP #(M-RC-R) 30 35 40 45 50 55 60 65 70 75

VG8L1 7 65.7 88.5 105 133 145 140 112 NG4L4 1 113 136 156 179 191 197 174 AVERAGE 89.1 112 130 156 168 169 143 STANDARD ERROR 23.4 23.9 25.6 23.2 23.3 28.5 31.1

Table 1 shows the weight progress of male sHW rats starting at thirty days from birth until life. These sHW rats were on a regular diet and ran five days a week at 15.0m/s for thirty minutes. Table 2

GROUP #(M-RC-NR) 30 35 40 45 50 55 60 65 70 75

NG5L2 11 91.7 132 149 167 189 191 OG5L5 7 86.1 162 178 207 263 263 280 285 AVERAGE 86.1 91.7 147 164 187 226 227 280 285 STANDARD ERROR 14.8 14.6 20 37.1 36

Table 2 shows the weight progress of male sHW rats starting at thirty days from birth until life. These sHW rats were non-‐runners on a regular diet.


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DISCUSSION

Possible neuroprotective effects were evaluated as the rats began their treadmill regimes. Research results showed no significant differences in body weight between the sHW runner and non-‐runner rats at the start of the trial. As they aged to be around 60 days old, there was an improvement in sustained body weight in the running group in comparison to the non-‐runner mutant sibling. Previous researches had shown sHW mutant rats to have an average lifespan of 63.2±1.9 days. Through exercise treatments the lifespan of the sHW rats statically increased by 13%, surviving an average of 70.4±2.1 days. There was a significant increase in their motor activity and weights after 50-‐55days of age. With future research, we can try to get a better understanding of the molecular mechanisms and pathways that mediate exercise induced neuroprotection, resulting treadmill exercise to the progressive loss of Purkinje cells and successive motor skills.

REFERENCES

Uhlendorf, T.L., et al., Neuroprotective effects of moderate aerobic exercise on the spastic Han– Wistar rat, a model of ataxia, Brain Res. (2010), doi:10.1016/j.brainres.2010.10.094

ACKNOWLEDGEMENTS I want to thank Dr. Randy Cohen for the amazing opportunity and allowing me to join, participate and be a part of his research. Also, I would like to thank Toni Uhlendorf and Shahab Younesi for all their constant support, guidance and aid in making sure I understand why and what I am doing. I would lastly like to thank Professor Mike Reynolds for allowing me to participate in this summer internship and opening up many opportunities for me.


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The Extraction and Identification of Wild Nematodes Through the Use of the Small Subunit

18 (SSU 18) gene Veronica Guizar, Sponsored by Professor Ray Hong, Department of Biology, California State University, Northridge, 18111 Nordhoff Street, Northridge, California 91330

INTRODUCTION Scarab Beetles are a diverse family of Beetles that are located in many parts of the world. Many begin to emerge around the month of June after dusk. Different species of nematodes are known to display necromenic interactions with these beetles, including Pristionchius Pacificus. P. Pacificus lives inside the live beetle in a dauer stage (a type of hibernating state), waiting for the insect to die in order to emerge and feed on the bacteria provided by the dead beetle. In this experiment, Cyclocephala Hirta Cyclocephala Pasadena beetles were cut open in order to extract different species of nematodes. These nematodes were then identified through the sequencing of the Small Subunit rDNA (SSU 18 gene). The SSU 18 gene has revolutionized the world of Nematode phylogeny, providing a more accurate phylogenic history than morphological characterization had provided before. According to Paul de Ley, Professor of Department of Nematology at UCR, there are 600 nematode species that have been identified based on SSU rDNA sequences.1 The SSU 18 gene is a reliable molecular marker due to its orthologous nature, which allows for comparison of different nematode sequences.

MATERIALS AND METHODS

Obtaining Wild Worms Cut open a scarab beetle in half onto an agar plate, parafilm the plate, and allow the beetle to sit for 2 weeks. Some beetles might yield nematodes.

1 “A Quick Tour of Nematode Diversity and the Backbone of Nematode Phylogeny.” Paul De Ley, Department of Nematology, University of California -‐ Riverside, Riverside, CA 92521, USA


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Courtesy of Dr. Hong Figure 1. These beetles were cut open in order to extract the nematodes from their hosts. Obtaining a Molecular Marker from Nematodes: Small Ribosomal RNA Gene (SSU, 18S) Isolation of Genomic DNA Pick a single worm obtained from the wild into 3 µl of single worm lysis buffer containing Proteinase-‐K into a PCR tube. Heat in PCR machine at 65°C for 1 hour, 95°C for 10 minutes and then allow it to cool to room temperature. PCR: Apex Mix:

• 2mM dNTPs (1.5µl) • 10x Taq Buffer (2µl) • 5 U/uL Taq polymerase (0.2µl) • H20

Prepare a master mix that can divide into 18 µl samples containing:

• 10 µl Apex mix • 1 µl RH 5401 primer • 1 µl RH 5402 reverse primer • 6 µl deionized H20


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Then add the 2 µl of the wild worm DNA into the appropriate PCR tube. Run the PCR reaction for 35 cycles at:

• 94°C – 3 minutes • 92°C – 45 seconds • 55°C – 45 seconds • 72°C – 1 minute 15 seconds • LAST EXTENSION: 72°C – 3 minutes

Gel Analysis: Make a 1% agarose gel using 50 ml of 1x TBE buffer and 0.5 grams of agarose. Heat solution in microwave for 1 minute 30 seconds, stirring every 30 seconds. Add 2 µl of ethidium bromide. Assemble the 75 ml bed, pour the solution, and allow it to solidify for 30 minutes. Insert bed into Gel Electrophoresis apparatus filled with 1x TBE buffer. Prepare each PCR sample by obtaining 5 µl of PCR product+ 3µl of gel loading solution and load each sample into its separate well. Run the gel at 100 V for 45 minutes. Then obtain your picture using a UV light. Expect ~900 bp PCR product band. Direct Sequencing of DNA fragment (~20 ng/ul) Add 2 volumes of DNA Binding Buffer for each sample. Set up centrifuge tubes with filter, and add sample directly to filter top. Centrifuge at the highest speed available for 1 minute. Add 200 µl of Wash buffer with Ethanol and centrifuge again at highest speed for 2 minutes. Place filters onto new tubes and add 15 µl of nanopure water, centrifuge at highest speed for 1 minute. Provide a 5 µl purified DNA sample + 1 µl of 10 mM RH5403 per sample for sequencing. Priming will start inside the PCR product, giving ~500 bp sequence. After sequencing, the results were blasted on the NCBI website in order to find the closest nucleotide sequence match.

RESULTS

Figure 2. 1% agarose gels showing PCR band products for nematodes 5,6,7,8,9, 10 and lab sample PS 312.


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Figure 3. 1% agarose gels showing PCR band products for nematodes 11, 12, 13.1, 14.1, 13.2, 15, 16.1, 16.2.

Figure 4. 1% agarose gels showing PCR band products for nematodes 18.1, 18.2 ,19.1, 20, 21.1, 21.2, 22.1, 23.1, 24, 25.


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Figure 5. 1% agarose gels showing PCR band products for nematodes 27.1,28, 30, 31, 32, 33.1, 33.2, 43.2, 45.1, 45.2, 46.1.

Figure 6. 1% agarose gels showing PCR band products for nematodes 30, 34.2, 35.1, 35.2, 36.1, 36.2, 37, 38, 39, 40, 34.2, 43.2, 44.2.


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WILD WORMS SEQUENCING RESULTS Species

Figure 7. This pie chart shows the percentages of how often of the 10 different species of nematodes occurred, of a total sample of 42 worms.


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Figure 8. Sequence alignment of 5 of the most common species found: Pristionchus Pacificus, Pristionchus Pseudaerivoris, Pristionchus Iheritieri, Diplogasteroides Magnus Strain, and Rhabditolaimus leukarti Strain. The differences in nucleotides are displayed by the different fluorescent colors.


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DISCUSSION Using single worm lysis, PCR, and sequencing, we were able to identify 10 different types of

nematodes in the beetles collected in 2012. The most common nematodes included Oscheius and Pristionchus Pacificus. One of the more interesting finds includes the Entomopathogenic nematode, Steinernema Carpocapsae. Another interesting find includes the nematode Rhabditolaimus Leuckarti Strain RS5525. When submitting the sequence into the NCBI blast program, we were not able to find a high percent match for the sequence. Included in this paper is a sequence alignment (Figure 8.) between the 5 most common nematodes found in our 2012 beetle collections. This alignment shows how a few nucleotide insertions/deletions in a nematode genome can change the species and how it interacts with its environment.

REFERENCES “A Quick Tour of Nematode Diversity and the Backbone of Nematode Phylogeny.” Paul De Ley, Department of Nematology, University of California -‐Riverside, Riverside, CA 92521, USA

ACKNOWLEDGEMENTS I would like to thank Professor Reynolds and Professor Brown for mentoring me and for helping me pursue this internship. I would specially like to thank Dr. Hong for his patience, guidance, and for allowing me the opportunity to work in his lab.


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Gene Therapy Using A Non-‐Viral Vector For Mucopolysaccharidosis Type 2

Eduardo Martin, Sponsored by Professors Aida Metzenberg and Stan Metzenberg, Department of Biology, California State University, Northridge, 18111 Nordhoff Street, Northridge, California 91330

INTRODUCTION

The role of a lysosome in a cell is to break down metabolites with the use of digestive enzymes. Currently, researchers have been able to identify over 50 different disorders of lysosomes. These disorders are known as lysosomal storage disorders (LSD) that result from defective enzymes within the cellular organelle. In the study of lysosomal storage disorders there exists families of LSDs. Mucopolysaccharidoses for example are a family of LSDs that are responsible for nine metabolic disorders. These nine disorders remain similar in that they prohibit the breaking down of certain sugars called glycoaminoglycans (GAGs), which are characterized by a repeating disaccharide unit. Of these nine disorders, only two, Mucopolysaccharidosis type one (MPS1) and Mucopolysaccharidosis type two (MPS2) are X-‐linked recessive. On the other hand, MPS types three through seven are autosomal recessive. MPS2 is commonly referred to as Hunter Syndrome and is distinguished by a low level or lack of Iduronate 2 Sulfatase (I2S). Further study of the I2S enzyme shows that it is essential in breaking down the GAGs dermatan sulfate and heparan sulfate by helping to enforce a cascade that is needed in breaking down GAGs. Due to this, a cell affected by a low level or lack of activity of the IDS gene results in the accumulation of GAGs, causing it to enlarge to the point of apoptosis. This leads researchers to believe that Hunter syndrome patients suffering from a lack or loss of I2S could therefore be provided with the IDS gene (which codes for I2S) in order to help cure them. When MPS2 patients underwent gene therapy by introducing the normal gene into their cells, a significant reduction of GAGs were found in their urine, as opposed to untreated patients. Because this process was done using a viral vector, there were very few successful outcomes. It is because the immune system automatically recognizes the harmless virus that the effectiveness of this approach is limited. The option now being researched with the help of new techniques is the usage of non-‐viral vectors to introduce the IDS gene into the patient. This would allow the vector to be inserted and transported throughout the body without the use of a virus. This would help in not having the immune system reject the vector being introduced into the cell. The non-‐viral vector being used for this experiment is called


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pEPito. This vector is 5245 base pairs long and contains roughly thirty restriction sites allowing flexibility for ligation.

MATERIALS AND METHODS

Competent Cells From XL1 Blue E.Coli Cells 1. Inoculate a single E.Coli colony into 5 ml of LB Broth from an agar plate labeled XL1 Blue. 2. Shake at 37 ºC and 50 rpm overnight. 3. After 24 hours, take 50 ml of LB Broth and pour into a 500 ml Erlenmeyer flask. 4. Add 50 ml of the overnight growth to the flask containing 50mL LB Broth. 5. Take a spectrophotometric reading at 600 nm at time zero. 6. Place flask in shaking incubator at 37 ºC and 50 rpm, making sure to not completely seal the flask

with a cap to allow for aeration. 7. Take spectrophotometric readings hourly until the absorbency reading is close to 0.6 at which

point the readings should be taken every 5 minutes with 600 uL aliquots. 8. Once the absorbency has reached 0.6 (or significantly close), divide the cells into two 50 ml falcon

tubes -‐ about 5 ml into each. 9. Centrifuge at 2000 xg for 5 minutes. 10. Discard the supernatant and add 5 ml of ice cold 0.1M CaCl to the pellet. 11. Gently dissolve the pellet and transfer all the contents into one of the two falcon tubes. 12. Centrifuge at 1000xg for 5 minutes. 13. Discard the supernatant and add 5 ml of cold 0.1M CaCl. 14. Add glycerol to 5% (add 80 uL of glycerol to falcon tube containing .5 ml of CaCl and pellet). 15. Divide competent cells into 0.1 ml aliquots in 0.5 ml tubes. 16. Store at -‐70 ºC.

Transformation of Competent Cells 1. Label two 15 ml tubes as “pEPito” and as “pUC19” and place both on ice. 2. Turn on the water bath to 42 ºC. Proceed to Step 3 only when at 42 ºC. 3. Thaw out 200 µl of competent cells and gently mix. 4. Place 100 µl of competent cells into each falcon tube. 5. Add 1 µl of pEPito (50pg/µl) to the first tube. Make sure to move the pipette through the cells

while dispensing the contents in a circular motion. Gently tap the falcon tube to mix. 6. Repeat step 3 with the control vector pUC19. 7. Place both tubes in ice for 30 minutes. 8. Place your SOC medium at room temperature. 9. Heat shock cells by placing tubes in a 42 ºC water bath for 45 seconds. Be very careful to not bump

the tubes. 10. Place tubes in ice for 2 minutes. 11. Add 900 uL of SOC medium to each of the falcon tubes. 12. Shake tubes at 37 ºC at 225 rpm for 1 hour. 13. Obtain 6 plates of LB Agar and label the plates: pEP A, pEP B, pEP C, pUC19 A, pUC19 B, pUC19. 14. Add the following to a sterilize the microtube: 25 µl of IPTG, 25 µl of X-‐Gal, 25 µl of Ampicillin. 15. Plating: Add your 75 µl solution from step 14 onto LB Agar Plate; Dip the glass spreader in 70%

ethanol; Flame the rod; Allow for the rod to cool for about 10-‐15 seconds; Spread the contents throughout the plates.

16. Let the plates dry for 15 minutes.


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17. Add 1 µl of Transformed cells to plate A. 18. Add 10 µl of Transformed cells to plate B. 19. Add the remainder of your transformed cells to plate C. 20. Repeat steps 16 – 18 for pUC19. 21. After 10 minutes, flip all plates labeled A and B. Allow plate C to dry longer before flipping. 22. Incubate overnight at 37 ºC. 23. After 24 hours, if white colonies present, take one single colony and place in 5mL of LB at 37C at

250rpm for 12 hours. 24. Also, take one colony and inoculate a slant by stabbing. 25. Perform Plasmid purification using Wizard® Plus SV Minipreps DNA Purification System.

X-‐GAL & IPTG 1. Spread 60 microliters of X-‐Gal & 60 microliters of IPTG solution onto an agar plate which contains

the pUC19 vector. 2. Place the plates in the incubator overnight to allow the bacterial colonies to grow.

Restriction Digestion Standard Reactants Volume (µL) 10X New England Buffer 4 (10 mg/ml(100X)) 2.0 uL 25ng/mL DNA 10.0 uL nH20 to 20uL 20.0 uL 5,000 unit/mL enzyme 0.2 uL

Thermocycler Conditions Temperature Time Number of Cycles 97.0 ºC 5 Seconds None 97.0 ºC 15 Seconds 10 55.0 ºC 15 Seconds 10 72.0 ºC 90 Seconds 10 97.0 ºC 15 Seconds 25 72.0 ºC 90 Seconds 25 72.0 ºC 5 minutes None 4.0 ºC Infinity None *Clean your area, discard pipette tips appropriately and put everything away.

Agarose Gel Electrophoresis 1. Weigh out agarose (a 2% gel -‐ 2.0 grams in 100mL of 50X TAE Buffer to be 1X final). 2. Microwave to dissolve agarose, being careful not to allow it to boil over. Let it cool until you can

touch the flask with your hands. 3. Cover the north and south ends of the gel mold using tape or press and seal. Pour the warm

agarose into the gel mold and allow to cool completely until it solidifies. 4. Assemble the electrophoresis apparatus and connect the electrodes to the power supply. 5. Remove the tape and/or the press and seal from the mold and place the mold on the

electrophoresis apparatus. 6. Run the gel at 100 volts for 1hour.


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7. Stain gel in a 1:1,000 dilution of 10mg/ml ethidium bromide stock solution for 10 minutes. Rinse gel in diH20.

8. Irradiate with UV light to reveal the DNA. 9. Photograph illuminated. Do not discard the gel until the experiment is finished. You may wrap the

gel in Saran Wrap and refrigerate to store, but note that over time, the contents of the gel will exit the gel by diffusion.

10. Clean your area, wash non-‐disposable glassware, autoclave trash as appropriate, autoclave clean glassware as appropriate.

RESULTS

Figure 1. Plates containing E.coli (XL1 Blue) cells with the pEPito vector. This picture shows the petri dishes after being taken out from an overnight wait in the incubator at 37 degrees Celsius. All plates from left to right are holding amplicillin, LB agar and pEPito containing E.coli cells spread on top. Left plate: 10 uL of E.coli cells. Middle plate: 100 uL of E.coli cells. Right plate: Left over amount/rest of E.coli cells. These same amounts were spread on the control plates that contained the same amount of cells, but the different pUC19 vector.


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Figure 2. DNA ladder ran along side digested pEPito plasmid in an agarose gel.

Figure two shows the results of the 1% agarose gel which was made run at 90 volts for 1.5 hours with the contents holding pEPito after being treated with Wizard Plus SV Minipreps DNA Purification System. First lane contents: gene ruler DNA ladder. Second lane contents: empty. Third lane contents: undigested pEPito vector (Results: ~1000 bp and ~3500 bp). Fourth lane contents: empty. Fifth lane contents: pEPito vector digested at two restriction sites: Apa1 & Sfi1 (Results: ~1000 bp and ~3500 bp). Sixth lane contents: empty. Seventh lane contents: pEPito vector digested at one restriction site: Apa1 (Results: ~1000 bp and ~3500 bp). Eight lane contents: empty. Ninth lane contents: pEPito vector digested at one restriction site: Sfi1 (Results: ~1000 bp and ~3500 bp).


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Figure 3. Gene Ruler This is the DNA ladder which was put into the agarose gel in order to compare our results to a definite amount of base pairs.

DISCUSSION The portion of the project discussed at hand revolved around transformation followed by a gel electrophoresis for the purpose of making excess amounts of the vector called pEPito. The first step in this process was to make competent cells. Once made, two trials of transformation were ran with these cells; the first trial called for a set of competent cells taking up the pEPito vector while the other trail had a set of competent cells take up a vector called pUC19. The reason for working with the pUC19 vector at this time was to use it as a control to see how competent the cells actually were. Due to the known high efficiency of transformation with the pUC19 vector, it was chosen to act as the control. To make sure the cells did indeed accept the pUC19 vector, reagent IPTG and organic compound X-‐GAL were introduced to the sample. Because the pUC19 vector contains a lac operon, IPTG was used to mimic the actions of lactose, which attached to the repressor causing it to release from the operon and therefore allowing gene expression of the operon. One of the two enzymes made from this gene expression is called beta-‐galactosidase. Beta-‐galactosidase interacted with X-‐GAL to make a blue and white byproduct


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released from the cell, therefore showing the signs of a successful transformation (No Figure -‐ Data not shown). Upon seeing the formations of blue and white colonies in the sample used, it was assured that the competent cells took up the pUC19 vector. In order to test for a successful transformation with the pEPito vector, the antibiotic ampicillin was introduced to the cells. Because the pEPito vector has an ampicillin resistance gene, the mere growth of colonies was enough to ensure that the cells had successfully taken up the vector (Figure 1 – Plates containing E.coli (XL1 Blue) cells with the pEPito vector.). The control also served useful to show the experiment to be a success up to this point. The wanted pEPito plasmid was then isolated using Wizard Plus SV Minipreps DNA Purification System. This purification kit killed off the bacteria (E.Coli XL1 Blue) while only leaving the plasmid behind. By checking the kit results with the nanodrop machine, the sample was assured to have optimal plasmid concentration (data not shown: 235ng/uL w/ 1.86260/280 ratio). To further verify results of the transformation and to make sure it was the vector that the nanodrop was reading and not foreign substances (i.e. left over pieces of cells from the clean up kit), it was decided that the isolated vector would be introduced to digestion. The vector was cut inside the thermocycler using the restriction sites/enzymes Sfi1 (found on 76 bp) and Apa1 (found on 4,053 bp). Unfortunately, the bands of the gel came out quite misleading (Figure 2 – DNA ladder ran along side digested pEPito plasmid in an agarose gel). The third lane, which had no digestion, showed a double digestion with contents that matched ~1000 bp and ~3500 bp. For the fifth lane that contained a double digestion, bands matched that of base pair lengths ~1000 bp and ~3500 bp long. The correct results from the double digest should have been two bands; one that lined up with ~3977 bp and the other at ~1268 bp. For the seventh lane Apa1 single digestion, the markers once again showed a double digestion with bands lining up at two places ~1000 bp and ~3500 bp long. There should have only been one marker that lined up at ~5200 bp. Lastly, in the lane nine Sfi1 single digestion, contents show a double digestion that lines up at ~1000 bp and ~3500 bp. Once again, there should have only been one marker that lined up at ~5200 bp. The fact that the digested pieces of DNA did not line up with their appropriate markers is likely due to a super coil in the plasmids from the samples used. This super coiled nature could have been produced because of the purification kit which unfortunately has a tendency to super coil plasmids. Regarding the reason that there were two digestions of the vector in all the lanes could only be accounted for by either contaminated samples or human error by accidental addition of a second restriction enzyme. Even though the correct base pair values were not obtained after restriction digestion, it should be noted that it does not necessarily mean that the vector pEPito was never in the E.Coli XL1 Blue colonies. On the contrary, it is a fact that the vectors were taken up by the cells through transformation. This is known because the colonies were able to grow after being introduced to the antibiotic ampicillin; something that would have been impossible had the colonies not taken up the vector. The problem this experiment faced was merely extracting the vector from the cells and running it through a digestion because of its highly probable super coiled nature. Most of our complications were found when trying to get out cells to perform transformation. For one reason or another, the E.coli cells were not picking up the pEPito plasmid. Only after many trials did we finally get one plate that was a success. A speculated reason for vast amounts of failure that was faced during this process was that perhaps the vector was too big at 5245 bp. Because this vector has shown


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to be difficult to work with overall, it is safe to call the pEPito plasmid an inefficient vector. For now, this project and gene therapy in general is being held back in a big part due to the slow advancement of efficient non viral vectors.

REFERENCES

Muenzer J. The mucopolysaccharidoses: a heterogeneous group of disorders with variable pediatric presentations. J Pediatr. 2004 May;144(5 Suppl):S27-‐34.

"IDS -‐ Iduronate 2-‐sulfatase -‐ Genetics Home Reference." Genetics Home Reference -‐ Your Guide to Understanding Genetic Conditions. 12 Dec. 2011. Web. 15 Dec. 2011.<http://ghr.nlm.nih.gov/gene/IDS>.

ACKNOWLEDGEMENTS

I would first and foremost like to thank Dr. Aida Metzenberg who graciously allowed me the opportunity to learn, work and understand how genetics is practiced in the lab. It is because of the sponsorship of both Dr. Stan and Aida Metzenberg that I have come to find a passion for the study and research of genetics, specifically gene therapy. I would also like to greatly thank Osvaldo Larios. As my instructor for this summer internship he patiently took the time every day to answer any questions I had and taught me all I have come to know in the lab. I also would like to say thank you to my fellow interns John Daniel David and Joyce Rivera who made going to lab every morning a fun and eventful experience. Lastly, I would like to thank and give my uttermost respect to my Biology teacher Professor Michael Reynolds for accepting me to be an intern in the TRAILS summer internship program. The guidance and teaching that you have bestowed on me throughout the spring and summer semesters has helped me reach places academically that I had only dreamed about. I will continue my academic career with all your advice in mind. Thank you so much.


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