Saumil+Jariwala's+Proposal



Addendum to iPS Project Due to budgetary issues, my research project could not be started at NCSU this summer. However, the research experience I garnered there has prompted me to propose an iPS-related project at NCSSM this year. One of the stated benefits of iPS research is that it is simple and inexpensive compared to other work with stem cells. The ease of the new process even prompted the VP of Stem Cell Research at Invitrogen to say in an interview, “it’s really easy—a high school lab can do it.” This statement has served as motivation for this project modification. By changing the genes I will be transfecting, changing the cell type to hair cells, and using equipment more easily available in a high school lab, it should be possible to create induced pluripotent stem cells in a high school lab. Instead of using HoxA10 and HoxB4, I propose using Oct4 and Sox2 with the chemical Valproic Acid, which has been shown to improve reprogramming efficiency. I will still be using the pMXs retroviral plasmid to create my retrovirus, although this retrovirus will now encode for Oct4, the linker peptide 2A, and Sox2. To make characterization of the reprogramming cells easier, I plan to obtain a lentivirus that fluoresces green in the presence of three proteins involved in iPS reprogramming: the Early Transposon II, Oct4, and Sox2. Instead of having to characterize through just doubling time and morphology, I will be able to demonstrate reprogramming through fluorescence as well. Instead of using RT-PCR to characterize my final cell product, I will use all three—doubling time, morphology, and fluorescence—to show that I have my final iPS product. While I have used many novel systems to reduce cost, I recognize that the cost of this project will still likely cost more than NCSSM will grant to this project. As a result, I plan to contact other labs working with iPS, cell culture, and molecular cloning to obtain some resources. I have included below a comprehensive literature review and proposal for this project modification. At the very end of the document, I have included a list of potential hazards and the protocols for many of the procedures I will be using in the lab. 

One of the major findings of the late 20th century was the discovery of stem cells, both embryonic and adult. These cells have the ability to self-renew and, more importantly, differentiate into cells of various lineages, from neurons to cardiomyocytes. However, stem cells of both types have limited applications. Although embryonic stem cells have the ability to differentiate into any kind of cell, ethical concerns have slowed down research on embryonic stem cells, and policymakers threaten future attempts at research. In contrast, there is less controversy with adult stem cells, but such cells are multipotent, and thus limited in differentiation capacity to specific sublineages. While debate over policy continued on, discoveries in 2006 and 2007 shifted debate and revealed an alternate cell type that possessed the benefits of embryonic stem cells without the controversy. [1] In 2007, two research teams reprogrammed adult fibroblasts into an embryonic stem cell-like state by transcription of four defined factors, chosen from c-Myc, Oct3/4, Sox2, Klf4, Nanog, and LIN28. [2] [3] These cells, deemed induced pluripotent stem cells (iPS), have a few advantages over embryonic stem cells. Besides being an ethical alternative, induced pluripotent stem cells have the advantage of being patient-specific: instead of attempting to match stem cell and patient based on HLA, or human leukocyte antigen, iPS can be generated from the patients themselves. In addition, the process is relatively simple, especially when compared to the very complicated alternate technique of somatic cell nuclear transfer. In fact, VP of Stem Cell Research at Invitrogen Mahendra Rao mentioned in an August 2008 interview with Scientific American that “’[iPS generation is] really easy—a high school lab can do it.’” [4] This statement poses an interesting challenge: is it possible to scale research on the forefront of molecular cloning to fit the confines of a high school laboratory? Successful replication of iPS induction experiments at the high school level involves a few major hurdles. Although iPS creation is technically simple, the cost of equipment and reagents can be prohibitively expensive. A list of the required equipment includes a laminar flow hood, a centrifuge, a thermocycler, an incubator shaker, an ultracentrifuge, an incubator, and a variety of expensive kits. In addition, extensive experience with embryonic stem cells (ESC) is often required in order to successfully identify the iPS. [5], [6] The focus of papers published on iPS heretofore has been largely on improving reprogramming efficiency and reducing the risk of mutagenesis. While increased efficiency may be necessary for the eventual transition of iPS to the clinic, this is not the case for the high school lab; theoretically, only a single iPS cell needs to be generated and isolated, as this stem cell can be expanded to larger populations. By selecting for reduction of cost and required expertise, it is possible to truly put iPS within the domain of the high school lab. In order to accomplish this goal, we propose the use of a variety of modifications to the standard protocol for iPS generation created by Takahashi of the Yamanaka lab. [7] iPS creation consists of a few general steps. The first is the introduction of a reporter construct that contains the genetic code for a fluorescent protein and resistance to a specific antibiotic into the cells to be reprogrammed. The promoter for a specific ES-expressing gene—such as Fbx15, Oct4, or Nanog—is attached to these two genes, and successful activation of this gene results in the expression of those two. While technically optional, this step both allows easy identification of iPS cells through fluorescence and also selection for pluripotency by antibiotic resistance. Although using a reporter would make identification simpler, creating cells with this reporter construct is difficult and is not possible at the high school level. However, without the presence of a reporter construct, considerable ES expertise is required to identify iPS morphologically. [8] Recent advances have opened up an alternative option to the reporter cassette. Hotta et. al recently generated a lentivirus that, upon activation of the Early Transposon and in the presence of Oct4 and Sox2, creates GFP and confers puromycin (puro) resistance. Instead of having to stably integrate a reporter into the desired cell line, the virus can simply be transfected. [9] To ensure safety, the virus is derived from a PL-SIN Lentivirus, which has a major deletion in the 3’ untranslated region (UTR). This deletion in the UTR prevents the replication of the packaging signal ψ, resulting in a lentivirus that cannot replicate. [10] In addition, the lentivirus extinguishes expression upon differentiation, which is useful when confirming a pluripotent state by differentiation assays. [11] Subsequently following is the placement of the desired transcription factors—usually Oct4, Sox2, Klf4, and c-Myc (the standard Yamanaka factors)—into a vector type, generally a retrovirus. This is usually a very involved process that involves a number of steps and long periods of waiting. We propose the use of three novel cloning mechanisms: the use of 2A self-cleaving peptides to create a bicistronic vector, creation of the plasmid through sequence and ligation independent cloning with T5 Exonuclease (SLIC), and the amplification of plasmid DNA through rolling circle amplification. These three in conjunction will allow the creation of a single plasmid that would contain the genes for all the desired transcription factors; that plasmid will be constructed in a single cloning step and without the need for bacterial transformation or succeeding minipreparation. When the gene for the 2A self-cleaving peptide is placed between the desired genes in a plasmid, the trigger of the plasmid’s promoter produces a long, single protein with all the desired amino acids of the individual proteins in it. 2A then acts to cleave itself out of the larger protein, resulting in relatively equal amounts of the desired proteins, now separated. It has been shown that using the 2A peptide results in the production of an extra lysine on the end of the desired products, but studies by the pioneering group that discovered this new method suggest that this has virtually no effect on the products. [12] Recently, Carey et. al used the 2A peptide to produce iPS cells from a single polycistronic virus with four factors. [13] SLIC cloning with T5 Exonuclease allows the ligation of multiple inserts and a cut vector through homologous recombination with treatment at a single temperature for sixty minutes. This novel approach works by chewing back the DNA with the exonuclease, ligating with Taq DNA Ligase, and then filling in the missing bases with Phusion DNA Polymerase, all in a single isothermal step. The use of SLIC replaces the laborious, multi-step process of cloning into a blunt-ended vector (such as TOPO), bacterial transformation and minipreparation, sequencing, digestion of the vector with restriction enzymes, and then cloning into the desired plasmid. [14], [15] A third revision that we propose to standard molecular cloning techniques is the use of rolling circle amplification instead of bacterial transformation and minipreparation. Although bacterial amplification of plasmids is a reliable and efficient technique, the process involves long waiting periods, DNA purification, and the use of incubation equipment that are atypical in high school labs. Instead, rolling circle amplification allows the geometric amplification of plasmid DNA by a PCR-like reaction that takes place at a single temperature. Specific DNA polymerases attach to random hexamer primers that attach to the desired plasmid. As the polymerase replicates across, old strands of DNA that are attached to the plasmid are knocked off by new strands; these strands that are knocked off are then free to be copied by other random hexamer primers, resulting in the geometric amplification of the plasmid DNA. [16], [17] The use of these three novel techniques will allow significant reductions to be made in both equipment and cost. The procedure for cellular induction to iPS has also been optimized for use in a high school lab. The standard procedure for iPS generation stems from Yamanaka’s original work on humans, where dermal fibroblasts with an embedded reporter were converted into iPS with retroviruses containing the genetic code for four transcription factors—Oct4, Sox2, Klf4, and c-Myc. Previously mentioned was the replacement of the endogenous reporter with the EOS lentivirus. In addition, a few other modifications have been made. Recent work has shown that two of the four transcription factors—Klf4 and c-Myc—are optional for conversion to iPS, and the two transcription factors have been either excluded or substituted by chemicals in some experiments. [18], [19] , [20] , [21] While the use of chemicals is considerably more simple than the creation of a retrovirus expressing a specific transcription factor, the chemical complement to all four factors has not yet been discovered, and some of these chemicals cost in excess of a thousand dollars. However, one chemical both replaces two of the required transcription factors and is rather inexpensive. Valproic acid (VPA), a histone deacetylase inhibitor, acts to substitute c-Myc, a very potent oncogene. [22] When c-Myc-induced iPS were injected into mice, tumors were produced in approximately 20%. [23] The use of low concentrations of VPA (e.g. 5 mM) radically improved the efficiency of reprogramming with the four original factors. [24] However, VPA used in conjunction with only Oct4 and Sox2 also produced iPS cells, albeit at a lower efficiency and slower rate. [25] Despite these detriments, we have opted to use this combination of just two factors and VPA, as it reduces the number of inserts that need to be cloned, allows the use of a bicistronic instead of polycistronic vector, and it avoids the use of the two oncogenic factors Klf4 and c-Myc. In addition to modifying the transcription factor cocktail, we also suggest the use of a nonstandard cell type and feeder layer. Dermal fibroblasts, while most prevalently used, must be obtained from skin biopsies and result in generally inefficient reprogramming. When keratinocytes were used with the standard Yamanaka factors, they produced iPS cell 100-fold more efficiently and twofold faster. In addition, keratinocytes are relatively easy to obtain: these cells can be obtained in large amounts from plucked hair follicles. [26], [27] Instead of culturing the keratinocytes on 3T3 cells and the iPS on mouse embryonic feeders (MEF), we also suggest the use of Matrigel, a chemical substitute. The culture of feeder cells is difficult, as specific media needs to be created for each cell type, and (more importantly) these cells need to be mitotically inactivated, which is generally done through either treatment with Mitomycin C (a potent mutagen) or gamma irradiation, both of which are unfeasible in a high school lab. [28], [29] In a supplementary experiment, Trond et. al used Matrigel to both culture the keratinocytes and keratinocyte-derived iPS. [30] The use of Matrigel would lessen the amount of time spent in cell culture, reduce the amount of culture media needed, and allow easy identification and isolation of iPS once reprogrammed. Our final proposed modification is not to the experimental design but rather to the way that the experiment Is performed. As the purpose of this experiment is to allow iPS creation at the high school level, it is important that other high schools be able to replicate our findings. While the publication of a comprehensive methods section is one way to do this, small errors in procedure can result in the failure of an entire experiment, especially when working with molecular cloning. As a result, we propose videotaping the entire process, from the initial molecular cloning to the final identification steps. Some online journals, such as the Journal of Visualized Experiments (JoVE), allow the publication of video experiments to the web. While some iPS generation protocols are already available in video format, the successful completion of this experiment would result in the first available protocol on “second-generation” iPS creation—the creation of iPS using some of the newer methods now available. By videotaping our experiment and distributing it to interested persons, we can help facilitate the spread of iPS creation to other high schools across the country. The arrival of pluripotent stem cells at the high school level would open up significant new opportunities for student learning. iPS generation involves some of the most crucial techniques in molecular cloning and cell culture. By involving students in the process, a high school could give students a leg up in both seeing how science is truly practiced and learning the vital skills necessary in pursuing a career in the biological sciences. In addition, this presents a remarkable opportunity to excite students in the sciences: granting students the gratification of being involved in two cutting-edge fields that possess considerable media hype—molecular cloning and stem cell research---will help foster the very excitement, curiosity, and mystery that drives scientists to make new discoveries. Stem cells will no longer be the things elusively mentioned in science textbooks and on TV but rather a staple of the classroom. Teachers will be able to teach cell concepts with a live model in the classroom: as pluripotent stem cells can differentiate into all cell types, teachers could potentially generate live models of all cell types in the classroom by following simple and established protocols. Inexpensive and simple iPS generation at the high school level presents myriad exciting new teaching opportunities.
 * Literature Review**

Works Cited Aasen, Trond, Angel Raya, Maria J Barrero, Elena Garreta, Antonella Consiglio, Federico Gonzalez, Rita Vassena, et al. “Efficient and rapid generation of induced pluripotent stem cells from human keratinocytes.” //Nat Biotech// 26, no. 11 (November 2008): 1276-1284. Carey, Bryce W., Styliani Markoulaki, Jacob Hanna, Kris Saha, Qing Gao, Maisam Mitalipova, and Rudolf Jaenisch. “Reprogramming of murine and human somatic cells using a single polycistronic vector.” //Proceedings of the National Academy of Sciences// 106, no. 1 (January 6, 2009): 157-162. Chen, LingYi, and Lin Liu. “Current progress and prospects of induced pluripotent stem cells.” //Science in China Series C: Life Sciences// 52, no. 7 (July 1, 2009): 622-636. Dupal, Mark. “Templiphi Presentation” presented at the DNA Sequencing Workshop at the John Curtin School of Medical Research's Biomolecular Resource Facility, Canberra, Australia, June 2002. http://brf.jcs.anu.edu.au/services/DNAsequencing/Templiphi.html. de Felipe, Pablo. “Skipping the co-expression problem: the new 2A "CHYSEL" technology.” //Genetic Vaccines and Therapy// 2, no. 1 (2004): 13. Feng, Bo, Jia-Hui Ng, Jian-Chien Dominic Heng, and Huck-Hui Ng. “Molecules that Promote or Enhance Reprogramming of Somatic Cells to Induced Pluripotent Stem Cells.” //Cell Stem Cell// 4, no. 4 (April 3, 2009): 301-312. Gibson, Daniel G, Lei Young, Ray-Yuan Chuang, J Craig Venter, Clyde A Hutchison, and Hamilton O Smith. “Enzymatic assembly of DNA molecules up to several hundred kilobases.” //Nat Meth// 6, no. 5 (May 2009): 343-345. Hotta, Akitsu, Aaron Y L Cheung, Natalie Farra, Kausalia Vijayaragavan, Cheryle A Seguin, Jonathan S Draper, Peter Pasceri, et al. “Isolation of human iPS cells using EOS lentiviral vectors to select for pluripotency.” //Nat Meth// 6, no. 5 (May 2009): 370-376. Huangfu, Danwei, Rene Maehr, Wenjun Guo, Astrid Eijkelenboom, Melinda Snitow, Alice E Chen, and Douglas A Melton. “Induction of pluripotent stem cells by defined factors is greatly improved by small-molecule compounds.” //Nat Biotech// 26, no. 7 (July 2008): 795-797. Huangfu, Danwei, Kenji Osafune, Rene Maehr, Wenjun Guo, Astrid Eijkelenboom, Shuibing Chen, Whitney Muhlestein, and Douglas A Melton. “Induction of pluripotent stem cells from primary human fibroblasts with only Oct4 and Sox2.” //Nat Biotech// 26, no. 11 (November 2008): 1269-1275. Jaenisch, Rudolf, and Richard Young. “Stem Cells, the Molecular Circuitry of Pluripotency and Nuclear Reprogramming.” //Cell// 132, no. 4 (February 2008): 567-582. Lehrman, Sally. “Dolly's Creator Moves Away from Cloning and Embryonic Stem Cells.” //Scientific American//, August 2008. http://www.scientificamerican.com/article.cfm?id=no-more-cloning-around. Li, Mamie Z, and Stephen J Elledge. “Harnessing homologous recombination in vitro to generate recombinant DNA via SLIC.” //Nat Meth// 4, no. 3 (March 2007): 251-256. Limat, Alain, and Friedrich K Noser. “Serial Cultivation of Single Keratinocytes from the Outer Root Sheath of Human Scalp Hair Follicles.” //J Investig Dermatol// 87, no. 4 (October 1986): 485-488. Logan, Aaron C, Carolyn Lutzko, and Donald B Kohn. “Advances in lentiviral vector design for gene-modification of hematopoietic stem cells.” //Current Opinion in Biotechnology// 13, no. 5 (October 2002): 429-436. Lyssiotis, Costas A., Ruth K. Foreman, Judith Staerk, Michael Garcia, Divya Mathur, Styliani Markoulaki, Jacob Hanna, et al. “Reprogramming of murine fibroblasts to induced pluripotent stem cells with chemical complementation of Klf4.” //Proceedings of the National Academy of Sciences// 106, no. 22 (June 2, 2009): 8912-8917. Maherali, Nimet, and Konrad Hochedlinger. “Guidelines and Techniques for the Generation of Induced Pluripotent Stem Cells.” //Cell Stem Cell// 3, no. 6 (December 4, 2008): 595-605. Muller, Lars UW, George Q Daley, and David A Williams. “Upping the Ante: Recent Advances in Direct Reprogramming.” //Mol Ther// 17, no. 6 (March 31, 2009): 947-953. Park, In-Hyun, Paul H Lerou, Rui Zhao, Hongguang Huo, and George Q Daley. “Generation of human-induced pluripotent stem cells.” //Nat. Protocols// 3, no. 7 (June 2008): 1180-1186. Reagin, Michael J., Theresa L. Giesler, Alia L. Merla, Jeanine M. Resetar-Gerke, Kinga M. Kapolka, and J. Anthony Mamone. “TempliPhi: A Sequencing Template Preparation Procedure That Eliminates Overnight Cultures and DNA Purification.” //Journal of Biomolecular Techniques : JBT// 14, no. 2 (June 2003): 143–148. Sambrook, Joseph, and David William Russell. //Molecular Cloning: A Laboratory Manual//. Third. Cold Spring Harbor, New York: Cold Springs Harbor Laboratory Press, 2001. Sayandip Mukherjee. “Induced Pluripotent Stem Cells: A New Hope or a New Controversy?.” //Opticon// 1826, no. 5 (2008). http://www.ucl.ac.uk/opticon1826/archive/Issue5/Article_BM_Mukherjee.pdf. Takahashi, Kazutoshi, Keisuke Okita, Masato Nakagawa, and Shinya Yamanaka. “Induction of pluripotent stem cells from fibroblast cultures.” //Nat. Protocols// 2, no. 12 (November 2007): 3081-3089. Takahashi, Kazutoshi, Koji Tanabe, Mari Ohnuki, Megumi Narita, Tomoko Ichisaka, Kiichiro Tomoda, and Shinya Yamanaka. “Induction of pluripotent stem cells from adult human fibroblasts by defined factors.” //Cell// 131, no. 5 (November 30, 2007): 861-872.

(1) To optimize the iPS creation protocol for cost/equipment and not reprogramming efficiency (2) To prove the principle that iPS could be made in a high school lab. (3) To make stem cells a more viable tool for teaching in high school classrooms by allowing the creation of iPS in the high school lab. Retroviruses will be created that contain the gene for one of two human transcription factors—Oct4 and Sox2—both of which are involved in reprogramming cells to a pluripotent state. Combinations of the two viruses will be introduced into keratinocytes along with valproic acid (VPA), an HDAC inhibitor. During the reprogramming stage, transfection efficiency will be assessed by red fluorescence and expression of Oct4, Sox2, and Early Transposon will be determined by green fluorescence. Pluripotency will be assessed by doubling time, morphology, and alkaline phosphatase staining. The use of these novel methods should reduce cost and the amount of equipment needed. With careful planning and the proper methods, induced pluripotent stem cells can indeed be created in the high school lab. All of these changes have been done before, but this is the first paper that will use them in combination. Differences from the published iPS protocols [31], [32] are noted below. · Use of a combined cassette containing Oct4 and Sox2 linked by the 2A cleavage protein instead of separate plasmids with each gene. · The plasmid will be created through sequence and ligation independent cloning (SLIC) · Instead of bacterial minipreparation to produce large quantities of plasmid DNA, we will use Templiphi Amplification. · Use of the EOS lentivirus instead of an embedded/endogenous reporter construct. · Use of Oct4, Sox2, and the chemical VPA instead of Oct4, Sox2, Klf4, and c-Myc · Use of adult keratinocytes instead of adult/fetal fibroblasts · Cells will be cultured on Matrigel instead of mouse embryonic feeders or other feeder cells. · Each step will be videotaped, resulting in the only video protocol of “second-generation” iPS creation · **pMXs-RFP** to measure transfection efficiency (while this should probably be made in our lab to ensure consistent results, it would increase the amount of money, complexity, and time required. In addition, high school labs won’t need this if following this procedure). · **EOS Lentiviral Cassette** to measure Early Transposon/Oct4/Sox2 expression through green fluorescence. (This would be excessively difficult to make in our lab, and high school labs won’t need it if this procedure is successful. Using this lentivirus would give us a plethora of data we can analyze, and it also gives us a backup plan if our induction efficiency is too low: this lentivirus confers puromycin resistance to cells that are being reprogrammed, so we could use antibiotic selection to increase yield). · **pMXs Plasmid** to contain our insert DNA. The Kitamura lab, which produced this plasmid, seems pretty happy to gives this DNA away. · **Oct4 and Sox2 cDNA** to clone into our plasmids. We could amplify it in our lab but it would increase cost but approximately $150 - $250 and would be a considerable time hog. Please see alternate procedure for mRNA reverse-transcription in alternate protocol section. ·<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> **2A cDNA** to link our vector inserts. 2A serves as a self-cleaving protein that attaches two other genes together in a cloning vector. Using 2A gives us a good reason to clone by homologous recombination (SLIC), as this method is generally overkill with just a single insert. Using 2A should save us time with creating the retrovirus. ·<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> **Lentivirus/Retrovirus Production Materials** to create our infectious agents. This consists of the following: PLAT-GP cells, Virus Packaging Kit, Retrovirus/Lentivirus Packaging Plasmids. __Molecular Cloning__ 1.<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> PCR where Oct4 sense and antisense primers amplify Oct4 cDNA. 2.<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> PCR where Sox2 sense and antisense primers amplify Sox2 cDNA. 3.<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> PCR where 2A sense and antisense primers amplify 2A cDNA. 4.<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> Run a gel with all three cDNAs. Include template and primers as negative controls. 5.<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> PCR purify all three pieces of cDNA. 6.<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> PCR a reaction mixture of pMXs backbone with PacI and EcoRI enzymes. Cut the gel with a scalpel at the proper band. Run a gel extraction kit to isolate the cut pMXs vector. 7.<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> Use a UV Spectrophotometer to get DNA concentration of cut pMXS, Oct4, Sox2, 2A cDNAs. 8.<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> PCR a reaction mixture of Oct4, 2A, Sox2, and pMXS with T5 Exonuclease (SLIC cloning). 9.<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> Run a gel with the cloned plasmid. Cut the gel with a scalpel at the proper band. Run a gel extraction kit to isolate the cut pMXs vector. 10.<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> Use Templiphi amplification to generate more plasmid DNA. Send in pMXS-O2S for sequencing. Use a UV Spectrophotometer to get DNA concentration of pMXS-O2S. __Cell Culture / iPS Induction__ 11.<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> Pluck hair follicles from consenting subjects and culture primary keratinocytes for iPS generation. Split after eight days. 12.<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> Introduce EOS lentiviral vector into PLAT-GP. Harvest lentivirus-infected media 48 hours after infection. 13.<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> Three days following split of keratinocytes, replace MEF-conditioned hES medium with lentivirus-infected media. (Centrifuge virus into pellet and resuspend in hES medium). a.<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> Avoid infecting a single plate of keratinocytes, as this will serve as an iPS control for not using EOS lentivirus. 14.<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> Introduce pMXS into PLAT-GP, producing RTV_RFP and RTV_Sox2-2A-Oct4 15.<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> Ensure that RFP expression is present. Collect viral supernatant at 48 and 72 hours. 16.<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> Infect keratinocytes with pMXS-O2S two to four times over a 48-hours period using FuGeNe. Assess transfection efficiency by measuring expression of RFP. 17.<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> Following the 48 hours, replate cells on Matrigel with hES culture media that contains 0.5mM of VPA (This is considered day 0 post-infection). Replace media every two days, and remove VPA from hES culture media following day 10 (post-infection). 18.<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> Allow ked ratinocytes to reprogram, replacing media regularly. Check for GFP expression to measure reprogramming efficiency. In one cell line, supplement ES media with puromycin at day 17 post-infection to select for iPS marker expression. 19.<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> iPS should be fully reprogrammed around day 30 post-infection. Check doubling time and morphology to assess ES cell-like state. Check for RFP expression to measure retroviral silencing. Colonies should be picked after a month with a 2ul micropipette and expanded separately. 20.<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> Confirm iPS state by alkaline phosphatase staining. Checking for red fluorescence will reveal transfection efficiency in keratinocytes. Checking for green fluorescence should reveal Oct4, Sox2, and Early Transposon expression, demonstrating a general measurement for reprogramming efficiency and time. Doubling time of iPS is longer than for differentiated cells, between 18 to 24 hours. Morphology of iPS is similar to ES and quite distinct from differentiated cells. Presence of alkaline phosphatase will also be assessed: alkaline phosphatase is an enzyme that is expressed in ES and iPS cells and not in keratinocytes. Keratinocytes with RFP Retrovirus [Negative Control] Keratinocytes with RFP Retrovirus and EOS Lentivirus (Puro Selected) Keratinocytes with RFP Retrovirus and EOS Lentivirus (Puro Unselected) Keratinocytes with RFP and Oct4-2A-Sox2 (O2S) Retroviruses Keratinocytes with RFP and O2S Retroviruses and EOS Lentivirus (Puro Selected) Keratinocytes with RFP and O2S Retroviruses and EOS Lentivirus (Puro Unselected) ·<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> Fridge at 4OC ·<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> Freezer at -20OC ·<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> Laminar Flow Hood ·<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> Incubator at 37OC with 5% CO2 ·<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> UV Spectrophotometer ·<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> Centrifuge, preferably one that can use adaptors for different sized tubes. Centrifuge should be able to spin 1.5mL tubes. ·<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> PCR Machine ·<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> Gel Electrophoresis Machine ·<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> Dissecting Microscope ·<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> Machine that can distill water. ·<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> Machine for sterilization (autoclave substitute). o<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> If modified properly, soaking in 10% bleach is a possible alternative. ·<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> 1.5 mL tubes. ·<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> Pasteur pipettes (10mL) and bulbs ·<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> Micropipettes: 2.5ul, 20ul, 200ul, 1000ul. Pipette tips: 2.5ul, 20ul, 200ul, 1000ul ·<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> Glass cuvettes for spectroscopy ·<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> Gel extraction kit: these can also be used to PCR Purify, provided that the first buffer is not used ·<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> Templiphi amplification kit ·<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> FuGeNe Transfection Kit ·<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> EcoRI, PacI enzymes. ·<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> New England Biolabs Buffer #1 (for use with the enzymes) ·<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> Bovine Serum Albumin (for enzyme use) ·<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> Phusion Polymerase, Taq Ligase, T5 Exonuclease ·<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> 5X Isothermal Reaction Buffer for SLIC ·<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> Matrigel for cell culture ·<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> DMEM with 10% FBS ·<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> Irradiated MEF-conditioned ES cell medium ·<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> PLAT-GP cells, ·<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> Retrovirus/Lentivirus Packaging Kit ·<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> Puromycin, Streptomycin, Penicillin, ·<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> TAE for Gel Electrophoresis ·<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> SYBR Green or Carolina BLUTM As each lab produces iPS differently and uses different differentiation criteria, I have included a list of prices we will not have to spend in order to give a more accurate estimate of what we are saving: ·<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> 75% of the resources used in DNA manipulation ·<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> 25% of the resources used in plasmid construction ·<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> Removed entire bacterial incubation, cultivation, transformation, preparation sequence o<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> Cost of Incubator Shaker: $5,000+ o<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> Cost of Minipreparation Kit: $90 (20 Reactions) o<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> Cost of LB Agar Plates with Carbenicillin: $40 (20 plates) o<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> Cost of LB Broth: $50 (25 liters) o<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> Price of TempliphiTM Amplification Kit: $210 (100 Reactions) o<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> **Approximate Savings: $5,000** ·<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> Removed entire blunt-end cloning sequence o<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> Cost of Blunt-End Cloning Kit and TOP10 Competent E. Coli: $500 (20 Reactions) o<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> **Approximate Savings: $500** ·<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> Using general UV spectrophotometer/DNA concentration ladder instead of NanodropTM UV Spectrophotometer o<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> Cost of NanodropTM ND-1000: $9,000 o<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> Price of UV Spectrophotometer: $1,000 o<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> Price of DNA Concentration Ladders: $50 - 100 o<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> **Approximate Savings: $8,000** ·<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> Using disposable laminar flow hood instead of permanent one. o<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> Cost of Laminar Flow Hood: $6,000+ o<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> Price of Disposable Laminar Flow Hood: $___ o<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> **Approximate Savings:** ·<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> **Approximate Total Savings: $13,500+** PL-SIN Lentiviral Vector Backbone
 * Purpose**
 * Experimental Design**
 * Engineering Goal**
 * Differences from Standard iPS Procedure:**
 * Materials Obtained from Other Labs:**
 * Timeline**
 * Condensed Methods**
 * Data Analysis**
 * Different Control Groups**
 * Equipment Required**
 * Materials Required**
 * Estimated Cost Reduction:**
 * Vector/Virus Maps**:

EOS Lentiviral Cassette

Ethidium Bromide (and to a lesser degree SYBR Green) are mutagenic compounds that are important for gel electrophoresis. The human cells that are transformed could contain endogenous viruses that could theoretically incorporate into the researcher’s genome and cells. This is a risk that all projects with human cell culture must deal with. I will be working with **retroviruses and lentiviruses**. If these viruses reproduce and infect cells besides those, there could be the risk of insertational mutagenesis.
 * __ Biological Hazards __**
 * Solution:** Use Carolina BLUTM, a compound kept on the Biology floor that allows DNA staining without carcinogenic risk and without the need for a UV transilluminator (thereby reducing cost). If I do use SYBR Green (because Carolina BLUTM may not stain well) I will follow all the procedures currently used on the Biology floor.
 * Solution:** Most universities consider normal molecular cloning (BSL1+) safety procedures (covered cell flasks, use of gloves and personal lab protection equipment, cleaning all glassware with either an autoclave or 10% bleach solution, etc.) as ample to prevent this risk, but another solution would be for me to use my own cells. Any viruses that have incorporated into the genome of those cells will also be in my genome. Thus, if I use my own cells, I don’t have to worry about viral incorporation.
 * Solution:** This problem is being dealt with in a variety of ways. Both the lentivirus and the retroviruses being used require two to three other viral packaging plasmids before becoming active and capable of infecting cells. Once the virus is infectious, BSL2 safety procedures will be used, as mandated by the Intel forms: all work will occur in a laminar flow hood with personal protection equipment and all glassware will be treated with a 10% bleach solution and/or be autoclaved. The lentiviral and retroviral backbones themselves have also been modified for the sake of safety. The backbones of both the retrovirus and the lentivirus have been modified to make the viruses replication-incompetent. The retrovirus we are using is derived from the Moloney Murine (Mouse) Leukemia Virus or MoMLV (of course, we are using panotropic packaging plasmids so it can infect humans instead of mice) and are labeled pMX. These plasmids have a deleted gag (d-gag) sequence, which prevents the copy of the packaging signal Ψ during attempted virus replication. Thus, the pMX-derived retroviruses are replication-incompetent and can’t replicate. The EOS lentivirus is derived from a PL-SIN lentivirus, the structure of which is included below:

This lentivirus has a deletion in the 5’ untranslated region (UTR), which once again prevents the copy of the packaging symbol, here called Ψ+ (as it is a modified version of Ψ). This 3’ UTR deletion is called a SIN (self-inactivating) deletion and has two functions: it renders the EOS lentivirus replication-incompetent and also reduces the risk of insertational mutagenesis during genome incorporation. Scalpel to cut gels so that we can isolate the cut pMXS vector out after running gel electrophoresis. <span style="font-size: 11pt; line-height: 115%; font-family: "Calibri","sans-serif";">
 * __Minor Hazards__**
 * Solution:** Use gloves and work carefully

//Estimated Time: 30 minutes for reaction preparation, Overnight for PCR, 30 minutes for gel extraction// //Equipment// ·<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> PCR Machine ·<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> Gel Electrophoresis Machine ·<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> Gel Extraction Kit ·<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> Centrifuge //Reagents// ·<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> EcoRI Enzyme ·<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> PacI Enzyme ·<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> New England Biolabs Buffer #1 10X Concentrate ·<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> Bovine Serum Albumin //Procedure// 1.<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> Determine the amount of cut vector desired total. 2.<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> Use the following amounts per reaction/PCR tray well to calculate the amount of each reagent need in your Master Mix (multiply these amounts by the number of desired reactions) ddH2O: 11ul NEB1 10x: 2ul BSA: 1ul (Equivalent of 10ug) Diluted DNA: 5ul (Equivalent of 300ng/reaction or even up to 5ug) EcoRI: 0.5ul (Equivalent of 5 U) __PacI: 0.5ul (Equivalent of 5 U)__ 3.<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> Add all of the reagents __in the aforementioned order__ in a single tube. When adding the EcoRI and PacI, __work in the freezer__ to avoid denaturing any of the enzymes. 4.<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> Incubate the tube at 37OC for 8 hours and then inactivate at 80OC for 5 minutes. Afterwards, store at 4OC. 5.<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> Split the tube so less than 5ug of DNA is in each gel well. Run the gel and include an undigested sample of the DNA and a ladder. 6.<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> Once the gel run is complete, use a scalpel to remove the region with the desired products. __Spend as little time possible under UV light__, as UV light quickly disintegrates samples. 7.<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> Run gel extraction kit and follow protocol.
 * PROTOCOLS AND MATERIALS**
 * Double Digestion of pMXS with EcoRI and PacI and Subsequent Gel Extraction**
 * Master Mix: 20ul**

<span style="font-size: 11pt; line-height: 115%; font-family: "Calibri","sans-serif";">
 * Source:** Piedrahita lab

//Estimated Time: 1 hour for reagent preparation, 30 minutes for cloning preparation, 90 minutes for PCR to complete, 30 minutes for gel extraction// //Equipment// ·<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> PCR Machine ·<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> Gel Extraction Kit ·<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> Centrifuge //Reagents// ·<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> 5X isothermal reaction buffer (see recipe below) ·<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> T5 Exonuclease ·<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> Phusion DNA polymerase ·<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> Taq DNA Ligase
 * Preparation of T5 SLIC Buffers and Cloning of Desired Plasmids**

//Procedure//

1.<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> Calculate the amount of DNA needed per template (insert, vectors, etc.) by using the conversion factor 0.0167ng/bp DNA or 0.003 ng bp-1 µL-1. The final concentration should be 100ng/20µL, with each template being in equimolar amounts. 2.<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> Add 25ul of the desired DNA at equimolar concentrations to 75ul of the isothermal assembly buffer to an appropriate tube. Put approximately 20ul into five PCR tubes and incubate at 50OC for 60 minutes. Cool to 10OC. 3.<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> Gel extract desired plasmid. Run a gel extraction kit and follow protocol. 4.<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;">

** Index ** |||| ** Volume (µL) ** |||| ** [Final Conc.] ** |||| ** RxNs ** |||||||||| ** Master Mix (µL) ** ||||  ||||||   ||||   || 1 ||||  DNA (equimolar)**ζ** || 5 ||||   10-100ng**ζ** / 20µL  |||| 5 ||||||||||   25  |||| <span style="font-family: "Arial Narrow","sans-serif";">□ ||||||   ||||   || 2 ||||  Isothermal Gap & Nick Repair Buffer high or low (1.33X) || 15 ||||   1X  |||| 5 ||||||||||   75  |||| <span style="font-family: "Arial Narrow","sans-serif";">□ ||||||   ||||   || |||| **20** ||||   ||||   ||||||||||   ||||   ||||||   ||||   || ζ Add **0.0167ng/ bp** DNA; Similarly add **0.003 ng bp-1 µL-1** ||||   ||||   ||||||||   ||||||   ||
 * Master Mix Isothermal DNA Assembly RxN** ||||  ||||||||||   ||||||   ||||||   ||   ||
 * Master Mix Isothermal DNA Assembly RxN** ||||  ||||||||||   ||||||   ||||||   ||   ||
 * Master Mix Isothermal DNA Assembly RxN** ||||  ||||||||||   ||||||   ||||||   ||   ||
 * Master Mix Isothermal DNA Assembly RxN** ||||  ||||||||||   ||||||   ||||||   ||   ||
 * Components ** ||
 * Total** ||

<span style="font-size: 11pt; line-height: 115%; font-family: "Calibri","sans-serif";">

**50˚C** ||  || **60 min** || || || **10˚C** || **∞** || //Recipe// 1.<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> Prepare six milliliters (6000 ul) of 5X isothermal reaction buffer by combining ·<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> 3 ml of 1 M Tris-HCl (pH 7.5) ·<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> 150 ul of 2 M MgCl2 ·<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> 60 ul of 100 mM dGTP ·<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> 60 ul of 100 mM dATP ·<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> 60 ul of 100 mM dTTP ·<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> 60 ul of 100 mM dCTP ·<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> 300 ul of 1 M DTT ·<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> 1.5 g PEG-8000 ·<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> 300 ul of 100 mM NAD
 * Cycling Conditions:** ||
 * Cycling Conditions:** ||

This buffer can be aliquoted and stored at –20oC.

2.<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> Prepare an assembly master mix by combining ·<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> 320 ul 5X isothermal reaction buffer ·<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> 0.64 ul of 10 U ul–1 T5 Exonuclease ·<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> 20 ul of 2 U ul–1 Phusion DNA polymerase ·<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> 160 ul of 40 U ul–1 Taq DNA ligase ·<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> dH2O up to a final volume of 1.2 ml

Fifteen microliters of this reagent-enzyme mix were aliquoted and stored at –20oC. This mixture can tolerate numerous freeze-thaw cycles and remains stable even after one year. The exonuclease amount is ideal for the assembly of DNA molecules with 20 – 150 bp overlaps.

Frozen 15 ul assembly mixture aliquots were thawed and then kept on ice until ready to be used.

5 ul of the DNA to be assembled was added to the master mixture in equimolar amounts. Between 10 and 100 ng of each 6 kb DNA fragment was added. For larger DNA segments, proportional amounts of DNA were added (for example, 250 ng of each 150 kb DNA segment). Incubations were performed at 50oC for 15 to 60 min (60 min was optimal).

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 * Source: **[33]** **

//Estimated Time: 30 minutes involvement, 4 – 18 hours incubation, 30 minutes for purification, 15 minutes for UV spec analysis// //Equipment// ·<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> Templiphi Amplification Kit ·<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> PCR Machine ·<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> UV Spectrophotometer ·<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> PCR Purification Kit ·<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> Centrifuge //Reagents// ·<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> A single PCR tube / small microfuge tube able to contain microliter amounts //Procedure// 1.<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> Transfer 5ul of sample buffer from amplification kit to a reaction tube. 2.<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> Transfer <0.5ul of purified plasmid DNA to the reaction tube (should contain 1pg – 10ng of DNA) 3.<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> Denature the sample. After the sample is added to sample buffer, seal the reaction tubes with an appropriate lid. Heat at 95 ˚C for 3 minutes, and then cool to room temperature or 4˚C. The rate of cooling is not critical. 4.<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> Prepare TempliPhi premix. In a separate tube, combine 5 μl of reaction buffer and 0.2 μl enzyme mix for each TempliPhi reaction. It is convenient to make a master mix sufficient for the required number of TempliPhi reactions just prior to use. Once made, the master mix must be used the same day and not stored for future use. This premix contains all components necessary to generate nonspecific amplification product, and must be kept on ice until ready for use. 5.<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> Transfer 5ul of the TempliPhi premix to the cooled, denatured sample. 6.<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> Incubate at 30 ˚C for 4–18 h. Denature enzymes by heating at 65OC for 10 minutes. Cool to 4OC. NOTE: DNA will be viscous. Dilution may be necessary. 7.<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> Run a PCR Purification kit and follow protocol. 8.<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> Use a UV spectrophotometer to analyze DNA concentration. 9.<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> Send in plasmid for sequencing.
 * Protocol for Templiphi Amplification of Plasmids**

<span style="font-size: 11pt; line-height: 115%; font-family: "Calibri","sans-serif";">
 * Source:** Templiphi Amplification Kit Manual [34]

//Estimated Time:// //Equipment// ·<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> Scissors ·<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> 6-Well Plate ·<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> 10mL Pasteur pipettes and bulb //Reagents// ·<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> //Procedure// 1.<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> Place 2mL of prewarmed Matrigel in each well prior to culture. After 30 minutes in the laminar flow hood, aspirate media. 2.<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> Pluck hair follicles from the scalp and cut the bulk of the shaft off. 3.<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> Place the internal part of a single scalp hair in each well containing irradiated MEF-conditioned ES cell medium 4.<span style="font-family: "Times New Roman"; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"> Check microscope regularly to observe outgrowth of keratinocytes. By day 8, split the cells, removing the original hair follicle. Media should be replaced approximately every two days. **NOTE: I am contacting the lab that created the keratinocyte iPS and will find out a more complex procedure ASAP.**
 * Protocol for Primary Keratinocyte Culture for iPS Generation**
 * Source:** Piedrahita lab,

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