Name: an efficient in vitro transposition method by a transcriptionally regulated sleeping beauty system packaged into an integration defective lentiviral vector
Uploaded: 2018-01-12T13:00:00
Description: Watch this Scientific Journal Video about An Efficient In Vitro Transposition Method by a Transcriptionally Regulated Sleeping Beauty System Packaged into an Integration Defective Lentiviral Vector at

We thank Prof. Zappavigna, University of Modena and Reggio Emilia, Modena, Italy for providing us with the pCCL-PGKrtTA2S-M2 plasmid. We also acknowledge Prof Z. Ivics (Paul Ehlrich Institute, Langen, Germany) and Prof. Z. Izvak (Max Delbruck Center for Molecular Medicine, Berlin, Germany) for pCMVSB100X and pT2/BH plasmids. This work was supported by DEBRA international and Italian Ministry of University and Research.

1. Plasmids employed
NOTE: Plasmid pCCL-PGKrtTA2S-M2 was kindly provided by Prof. Zappavigna (University of Modena and Reggio Emilia, Modena, Italy). The pCMVSB100X plasmid carrying the coding sequence&#160;of hyperactive transposase and pT2/BH transposon plasmid were kindly provided by Prof Z. Ivics (Paul Ehrlich Institute, Langen, Germany) and Prof. Z. Izvak (Max Delbruck Center for Molecular Medicine, Berlin, Germany).
<ol>
	<li>For all cloning in Escherichia Coli (<em...

<ol>
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N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sleeping+beauty+system+to+redirect+T-cell+specificity+for+human+applications.">Sleeping beauty system to redirect T-cell specificity for human applications.</a> J Immunother. 36 (2), 112-123 (2013).</li><li>Johnen, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sleeping+Beauty+transposon-mediated+transfection+of+retinal+and+iris+pigment+epithelial+cells.">Sleeping Beauty transposon-mediated transfection of retinal and iris pigment epithelial cells.</a> Invest Ophthalmol Vis Sci. 53 (8), 4787-4796 (2012).</li><li>Koso, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transposon+mutagenesis+identifies+genes+that+transform+neural+stem+cells+into+glioma-initiating+cells.">Transposon mutagenesis identifies genes that transform neural stem cells into glioma-initiating cells.</a> Proc Natl Acad Sci U S A. 109 (44), E2998-E3007 (2012).</li><li>Belay, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+hyperactive+transposons+for+genetic+modification+of+induced+pluripotent+and+adult+stem+cells:+a+nonviral+paradigm+for+coaxed+differentiation.">Novel hyperactive transposons for genetic modification of induced pluripotent and adult stem cells: a nonviral paradigm for coaxed differentiation.</a> Stem Cells. 28 (10), 1760-1771 (2010).</li><li>Grabundzija, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sleeping+Beauty+transposon-based+system+for+cellular+reprogramming+and+targeted+gene+insertion+in+induced+pluripotent+stem+cells.">Sleeping Beauty transposon-based system for cellular reprogramming and targeted gene insertion in induced pluripotent stem cells.</a> Nucleic Acids Res. 41 (3), 1829-1847 (2013).</li><li>Kues, W. 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C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correction+of+Recessive+Dystrophic+Epidermolysis+Bullosa+by+Transposon-Mediated+Integration+of+COL7A1+in+Transplantable+Patient-Derived+Primary+Keratinocytes.">Correction of Recessive Dystrophic Epidermolysis Bullosa by Transposon-Mediated Integration of COL7A1 in Transplantable Patient-Derived Primary Keratinocytes.</a> J Invest Dermatol. 137 (4), 836-844 (2017).</li><li>Zhang, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integration+profile+and+safety+of+an+adenovirus+hybrid-vector+utilizing+hyperactive+sleeping+beauty+transposase+for+somatic+integration.">Integration profile and safety of an adenovirus hybrid-vector utilizing hyperactive sleeping beauty transposase for somatic integration.</a> PLoS One. 8 (10), e75344 (2013).</li><li>Zhang, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hybrid+adeno-associated+viral+vectors+utilizing+transposase-mediated+somatic+integration+for+stable+transgene+expression+in+human+cells.">Hybrid adeno-associated viral vectors utilizing transposase-mediated somatic integration for stable transgene expression in human cells.</a> PLoS One. 8 (10), e76771 (2013).</li><li>Turunen, T. A., Laakkonen, J. P., Alasaarela, L., Airenne, K. J., Yla-Herttuala, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sleeping+Beauty-baculovirus+hybrid+vectors+for+long-term+gene+expression+in+the+eye.">Sleeping Beauty-baculovirus hybrid vectors for long-term gene expression in the eye.</a> J Gene Med. 16 (1-2), 40-53 (2014).</li><li>Galla, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Avoiding+cytotoxicity+of+transposases+by+dose-controlled+mRNA+delivery.">Avoiding cytotoxicity of transposases by dose-controlled mRNA delivery.</a> Nucleic Acids Res. 39 (16), 7147-7160 (2011).</li><li>Moldt, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+genomic+integration+profiling+of+Sleeping+Beauty+transposons+mobilized+with+high+efficacy+from+integrase-defective+lentiviral+vectors+in+primary+human+cells.">Comparative genomic integration profiling of Sleeping Beauty transposons mobilized with high efficacy from integrase-defective lentiviral vectors in primary human cells.</a> Mol Ther. 19 (8), 1499-1510 (2011).</li><li>Recchia, A., Perani, L., Sartori, D., Olgiati, C., Mavilio, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Site-specific+integration+of+functional+transgenes+into+the+human+genome+by+adeno/AAV+hybrid+vectors.">Site-specific integration of functional transgenes into the human genome by adeno/AAV hybrid vectors.</a> Mol. Ther. 10, 660-670 (2004).</li><li>Urlinger, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploring+the+sequence+space+for+tetracycline-dependent+transcriptional+activators:+novel+mutations+yield+expanded+range+and+sensitivity.">Exploring the sequence space for tetracycline-dependent transcriptional activators: novel mutations yield expanded range and sensitivity.</a> Proc Natl Acad Sci U S A. 97 (14), 7963-7968 (2000).</li><li>Cocchiarella, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptionally+regulated+and+nontoxic+delivery+of+the+hyperactive+Sleeping+Beauty+Transposase.">Transcriptionally regulated and nontoxic delivery of the hyperactive Sleeping Beauty Transposase.</a> Mol Ther Methods Clin Dev. 3, 16038 (2016).</li><li>Yanez-Munoz, R. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effective+gene+therapy+with+nonintegrating+lentiviral+vectors.">Effective gene therapy with nonintegrating lentiviral vectors.</a> Nat Med. 12 (3), 348-353 (2006).</li><li>Dull, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+third-generation+lentivirus+vector+with+a+conditional+packaging+system.">A third-generation lentivirus vector with a conditional packaging system.</a> J Virol. 72 (11), 8463-8471 (1998).</li><li>Bak, R. O., Mikkelsen, J. 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Here, we describe a widely accessible methodology to stably integrate a GOI into the genome of target cells by Sleeping Beauty-mediated transposition. Although the SB system was developed to provide a nonviral method for genome editing, efficient delivery of the integration machinery (transposase and T2 transposon) is mandatory. Therefore, to use the SB system on hardly transfectable primary cells, a viral delivery of SB components was pursued in recent years10,<sup class="xref...

The hyperactive SB100X transposase coupled to the T2-based transposon has already been used in preclinical gene therapy applications1,2,3, genome modifications4,5, and induced pluripotent stem cell (iPSC) reprogramming6,7. Typically, the SB components are transiently provided by plasmid transfection and a strong constitutive promoter drives the expression of the transposase. However, despite the improved new methods of transfection, delivery of the two-component system remains a challenge for many applications. Thus, the development of a new delivery protocol has increasing interest. Besides transfection methods for plasmid8 and mRNA9, viral delivery based on adenoviral10,11, adeno-associated viral (AAV)12, Baculovirus13, gammaretroviral14 and lentiviral vectors15 has been proposed in the past. Notably, the system of choice must guarantee a transient delivery of the SB components without integration of the viral vector. Nevertheless, the constitutive expression of transposase raises safety concerns due to the risk of uncontrollable rehopping of the transposon.
Therefore, transcriptional regulation of SB100X by a refined Tet-ON system is the first challenge. A modified Tet-responsive promoter, obtained by substituting the original developed minimal cytomegalovirus (CMV) promoter with the minimal promoter (-81) of the Timidine Kinase (TK) gene of Herpes Simplex Virus-1 (HSV-1)16, is cloned upstream of the SB100X cDNA (pTetOTKSB100X). The mutated reverse-tetracycline-transactivator rtTA2s-M217 is expressed under the control of the strong constitutive promoter (phosphoglycerate promoter, PGK) cloned in a different plasmid (pCCL-PGKrtTA2S-M2). When added to the culture medium, dox binds the rtTA2s-M2 modulator and tethers the Tet promoter complex or, resulting in tightly regulated induction of SB100X expression18.
The second major challenge arises from the inefficient transfection of human primary cells by several physical and chemical methods. To efficiently deliver transposase in human cells resistant to transfection, the SB100X and the rtTA2s-M2 expression cassettes are packaged into two integration defective lentiviral vectors (IDLVs)19: IDLVTKSB and IDLVrtTA2s-M2. Viral vectors are transiently produced as third generation vectors in HEK293T cells20 and pseudotyped with the glycoprotein G of the Vescicular Stomatitis Virus (VSV-G), which confers a broad spectrum of infection. Vector particles are concentrated by ultracentrifugation and titrated by HIV-1 Gag p24 immunocapture. To transduce cell lines and primary cells, vector particles are complexed with polybrene and are incubated with target cells in the presence or absence of dox. Drug-dependent activation of SB100X expression in transduced cells is measured by quantitative RT-PCR (qRT-PCR)18.
Once Tet-regulated SB100X expression is demonstrated, transposition of the GOI (e.g., green fluorescent protein, GFP) into the target cell genome follows the molecular events clearly schematized by Bak and Mikkelsen21. A third IDLV vector carrying an expression cassette for the GOI cloned between the T2-transposon IRs (IDLVT2) has to be constructed and packaged as mentioned above. Lastly, the three IDLV vectors can be used to efficiently transduce human cells (e.g., primary keratinocytes) in vitro and integrate the GOI in the presence of doxycycline18.

<table>
	<tbody>
		<tr>
			<td>Dulbecco&#8217;s modified Eagle&#8217;s medium with 4.5g/L glucose w/o L-Glutamine</td>
			<td>Lonza</td>
			<td>BE12-614F</td>
			<td>Cell culture medium.</td>
		</tr>
		<tr>
			<td>HyClone Fetal Bovine Serum (U.S.), Characterized</td>
			<td>GE Healthcare Life Sciences</td>
			<td>SH30071.03</td>
			<td>Serum used HEK293T cell culture for lentiviral particle production.</td>
		</tr>
		<tr>
			<td>Penicillin 10.000 UI/ml Streptomycin 10.000 UI/ml</td>
			<td>Lonza</td>
			<td>DE17-602E</td>
			<td>Reagents for cell culture medium.</td>
		</tr>
		<tr>
			<td>L-Glutamine 200mM</td>
			<td>Lonza</td>
			<td>BE17-605E</td>
			<td>Reagent for cell culture medium.</td>
		</tr>
		<tr>
			<td>Sodium Chloride (NaCl)</td>
			<td>Sigma-Aldrich</td>
			<td>S7653</td>
			<td>Reagent for HBS 2X preparation.</td>
		</tr>
		<tr>
			<td>HEPES Buffer, 1M Stock in normal saline</td>
			<td>Lonza</td>
			<td>BE17-737E</td>
			<td>Reagent for HBS 2X preparation.</td>
		</tr>
		<tr>
			<td>Disodium hydrogen phosphate dihydrate (Na2HPO4&#183;2H2O), 99.5%</td>
			<td>Merck</td>
			<td>106580</td>
			<td>Reagent for HBS 2X preparation.</td>
		</tr>
		<tr>
			<td>Hydrochloric acid - ACS reagent 37% (HCl)</td>
			<td>Sigma-Aldrich</td>
			<td>320331</td>
			<td>Reagent for HBS 2X preparation.</td>
		</tr>
		<tr>
			<td>Sterile water for injection</td>
			<td>Fresenius Kabi</td>
			<td>-</td>
			<td>Reagent for HBS 2X preparation.</td>
		</tr>
		<tr>
			<td>0.45 &#181;m PESS filter</td>
			<td>Whatman</td>
			<td>10462100</td>
			<td>Filters used to remove cell debris from viral supernatant.</td>
		</tr>
		<tr>
			<td>Polyallomer Beckman tubes</td>
			<td>Beckman Coulter</td>
			<td>326823</td>
			<td>Tubes for concentrating IDLV vectors by ultracentrifugation.</td>
		</tr>
		<tr>
			<td>PBS-1X w/o Ca, Mg</td>
			<td>Lonza</td>
			<td>BE17-516F</td>
			<td>Buffer for cell wash and lentiviral particle resuspension.</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>Sigma-Aldrich</td>
			<td>A2153</td>
			<td>(Optional) Buffer for lentiviral particle resuspension.</td>
		</tr>
		<tr>
			<td>HIV-1 Gag p24 immunocapture kit</td>
			<td>Perkin Elmer</td>
			<td>NEK50001KT</td>
			<td>Kit for IDLV particle titration.</td>
		</tr>
		<tr>
			<td>Polybrene</td>
			<td>Sigma-Aldrich</td>
			<td>107689</td>
			<td>Reagent to enhance transduction efficiency.</td>
		</tr>
		<tr>
			<td>Trypsin (10X) 2.5% in HBSS w/o Ca, Mg</td>
			<td>GIBCO</td>
			<td>BE17-160E</td>
			<td>Reagent to detach HeLa cells (stock solution, to be diluted).</td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid disodium salt (EDTA)</td>
			<td>100938B</td>
			<td>AnalaR BDH</td>
			<td>Reagent for trypsin working solution preparation.</td>
		</tr>
		<tr>
			<td>0.5% Trypsin-EDTA, no phenol red (10X)</td>
			<td>GIBCO</td>
			<td>15400-054</td>
			<td>Reagent to detach keratinocytes (stock solution, to be diluted).</td>
		</tr>
		<tr>
			<td>Donor Bovine Serum, New Zealand Origin</td>
			<td>GIBCO</td>
			<td>16030-074</td>
			<td>Serum for Swiss mouse 3T3-J2 cell culture (feeder layer).</td>
		</tr>
		<tr>
			<td>Ham&#8217;s F12 media</td>
			<td>GIBCO</td>
			<td>21765</td>
			<td>Medium for keratinocytes.</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Lonza</td>
			<td>DE14-801F</td>
			<td>Serum for HeLa cell culture medium.</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum, qualified, Australia Origin</td>
			<td>GIBCO</td>
			<td>10099-141</td>
			<td>Serum for human primary keratinocyte culture medium.</td>
		</tr>
		<tr>
			<td>Insulin from bovine pancreas</td>
			<td>Sigma-Aldrich</td>
			<td>I5500</td>
			<td>Reagents for keratinocyte growth.</td>
		</tr>
		<tr>
			<td>Adenine</td>
			<td>VWR</td>
			<td>1152-25</td>
			<td>Reagents for keratinocyte growth.</td>
		</tr>
		<tr>
			<td>Hydrocortisone</td>
			<td>VWR</td>
			<td>3867-1</td>
			<td>Reagents for keratinocyte growth.</td>
		</tr>
		<tr>
			<td>Cholera Toxin from Vibrio cholerae</td>
			<td>Sigma-Aldrich</td>
			<td>C8052</td>
			<td>Reagents for keratinocyte growth.</td>
		</tr>
		<tr>
			<td>Triiodothyronine</td>
			<td>Sigma-Aldrich</td>
			<td>T5516</td>
			<td>Reagents for keratinocyte growth.</td>
		</tr>
		<tr>
			<td>Human EGF</td>
			<td>Austral Biological</td>
			<td>GF-010-9</td>
			<td>Reagents for keratinocyte growth.</td>
		</tr>
		<tr>
			<td>Mouse monoclonal APC-conjugated Anti-Feeder antibody</td>
			<td>Miltenyi Biotech</td>
			<td>130-096-099</td>
			<td>To label feeder layer.</td>
		</tr>
		<tr>
			<td>RNeasy Plus Mini Kit</td>
			<td>Qiagen</td>
			<td>74134</td>
			<td>RNA purification kit.</td>
		</tr>
		<tr>
			<td>SuperScript III Reverse Transcriptase</td>
			<td>Life Technologies</td>
			<td>18080051</td>
			<td>Reverse transcriptase kit.</td>
		</tr>
		<tr>
			<td>Deoxynucleotide Mix, 10 mM (dNTP)</td>
			<td>Sigma-Aldrich</td>
			<td>D7295</td>
			<td>Reagent for semi-quantitative PCR.</td>
		</tr>
		<tr>
			<td>UltraPure DNase/RNase-Free Distilled Water</td>
			<td>(any)</td>
			<td>-</td>
			<td>Reagent for semi-quantitative PCR and for qRT-PCR.</td>
		</tr>
		<tr>
			<td>GoTaq G2 Hot Start Polymerase</td>
			<td>Promega</td>
			<td>M740B</td>
			<td>Taq polymerase.</td>
		</tr>
		<tr>
			<td>Agarose, for molecular biology</td>
			<td>Sigma-Aldrich</td>
			<td>A9539</td>
			<td>Reagent for agarose gel electrophoresis.</td>
		</tr>
		<tr>
			<td>UltraPure TBE Buffer, 10X</td>
			<td>Invitrogen</td>
			<td>15581028</td>
			<td>Reagent for agarose gel electrophoresis, to be dilute (1X) in ddH2O.</td>
		</tr>
		<tr>
			<td>Ethidium bromide</td>
			<td>Sigma-Aldrich</td>
			<td>E1510</td>
			<td>Reagent for agarose gel electrophoresis.</td>
		</tr>
		<tr>
			<td>Doxycycline</td>
			<td>Sigma-Aldrich</td>
			<td>D-9891</td>
			<td>drug to induce transposase expression in HeLa cells and in keratinocytes</td>
		</tr>
		<tr>
			<td>TaqMan Universal PCR Master Mix</td>
			<td>Applied Biosystem</td>
			<td>4304437</td>
			<td>TaqMan Universal PCR Master Mix including primers and probe.</td>
		</tr>
		<tr>
			<td>15mL High Clarity polypropylene Centrifuge Tube, Conical Bottom, with Dome Seal Screw Cap, Sterile</td>
			<td>Falcon Corning</td>
			<td>352096</td>
			<td>Tubes for cell collection and vector dilution preparation.</td>
		</tr>
		<tr>
			<td>150 mm TC-Treated Cell Culture Dish with 20 mm Grid, Sterile</td>
			<td>Falcon Corning</td>
			<td>353025</td>
			<td>Cell culture dish for viral production.</td>
		</tr>
		<tr>
			<td>6 Well Clear Flat Bottom TC-Treated Multiwell Cell Culture Plate, with Lid, Individually Wrapped, Sterile</td>
			<td>Falcon Corning</td>
			<td>353046</td>
			<td>Cell culture plate for HeLa and human primary keratinocyte transduction.</td>
		</tr>
		<tr>
			<td>Safe-Lock microcentrifuge tubes, volume 1.5 mL</td>
			<td>Eppendorf</td>
			<td>0030 120.086</td>
			<td>Tubes for dilution preparation.</td>
		</tr>
		<tr>
			<td>Safe-Lock microcentrifuge tubes, volume 2 mL</td>
			<td>Eppendorf</td>
			<td>0030 120.094</td>
			<td>Tubes for dilution preparation.</td>
		</tr>
		<tr>
			<td>0.2ml PCR Tubes with flat caps, RNase, DNase, DNA and PCR Inhibitor free</td>
			<td>(any)</td>
			<td>-</td>
			<td>Tubes for semi-quantitative PCR.</td>
		</tr>
		<tr>
			<td>MicroAmp Fast Optical 96-Well Reaction Plate with Barcode, 0.1 mL</td>
			<td>Applied Biosystem</td>
			<td>4346906</td>
			<td>96-well for qRT-PCR.</td>
		</tr>
		<tr>
			<td>5mL Round Bottom Polystyrene Tube, without Cap</td>
			<td>BD Falcon</td>
			<td>352052</td>
			<td>Tubes for flow cytometry acquisition.</td>
		</tr>
		<tr>
			<td>Disposable Serological Pipets, Polystyrene, Individually Wrapped, Sterile (different volumes)</td>
			<td>(any)</td>
			<td>-</td>
			<td>Pipettes for sterile tissue culture applications.</td>
		</tr>
		<tr>
			<td>Filter Tips, Sterile, and RNase, DNase, DNA and Pyrogen free (different volumes)</td>
			<td>(any)</td>
			<td>-</td>
			<td>Filter tips for sterile tissue colture and molecular biology applications.</td>
		</tr>
		<tr>
			<td>pH meter</td>
			<td>(any)</td>
			<td>-</td>
			<td>Equipment for measurement of HBS 2X pH.</td>
		</tr>
		<tr>
			<td>PCR Thermal cycler</td>
			<td>(any)</td>
			<td>-</td>
			<td>Equipment for semi-quantitative PCR.</td>
		</tr>
		<tr>
			<td>Agarose gel electrophoresis equipment</td>
			<td>(any)</td>
			<td>-</td>
			<td>Equipment for agarose gel electrophoresis of semi-quantitative PCR.</td>
		</tr>
		<tr>
			<td>BD FACS Canto II 2LSR 4/2</td>
			<td>BD</td>
			<td>-</td>
			<td>Flow cytometer.</td>
		</tr>
		<tr>
			<td>7900HT Fast Real-Time PCR system</td>
			<td>Applied Biosystem</td>
			<td>7900HT</td>
			<td>Equipment for qRT-PCR.</td>
		</tr>
		<tr>
			<td>Benchtop centrifuges for efficient sample processing in cell culture applications, refrigerated, with microplate buckets.</td>
			<td>(any)</td>
			<td>-</td>
			<td>Centrifuge for cell culture processing and spinoculation.</td>
		</tr>
		<tr>
			<td>Optima L-90K Ultracentrifuge equipped with SW32TI rotor</td>
			<td>Beckman Coulter</td>
			<td>-</td>
			<td>Ultracentrifuge for lentiviral particle production.</td>
		</tr>
		<tr>
			<td colspan="4">Equipment for cell culture and molecular biology laboratory (e.g., Class II laminar flow hood designed for work involving BSL2 materials, cell culture incubator,refrigerators and freezers (+4 &#176;C, -20 &#176;C, -80 &#176;C), micropipettes and pipet-aid).</td>
		</tr>
		<tr>
			<td colspan="4">Protective laboratory coats, gloves, face protection in accordance with Institute rules and regulations, in particular for lentiviral particle production and transduction.</td>
		</tr>
	</tbody>
</table>

an efficient in vitro transposition method by a transcriptionally regulated sleeping beauty system packaged into an integration defective lentiviral vector

The Sleeping Beauty (SB) transposon is a non-viral integrating system with proven efficacy for gene transfer and functional genomics. To optimize the SB transposon machinery, a transcriptionally regulated hyperactive transposase (SB100X) and T2-based transposon are employed. Typically, the transposase and transposon are provided transiently by plasmid transfection and SB100X expression is driven by a constitutive promoter. Here, we describe an efficient method to deliver the SB components to human cells that are resistant to several physical and chemical transfection methods, to control SB100X expression and stably integrate a gene of interest (GOI) through a &#34;cut and paste&#34; SB mechanism. The expression of hyperactive transposase is tightly controlled by the Tet-ON system, widely used to control gene expression since 1992. The gene of interest is flanked by inverted repeats (IR) of the T2 transposon. Both SB components are packaged in integration defective lentiviral vectors transiently produced in HEK293T cells. Human cells, either cell lines or primary cells from human tissue, are in vitro transiently transduced with viral vectors. Upon addition of doxycycline (dox, tetracycline analog) into the culture medium, a fine-tuning of transposase expression is measured and results in a long-lasting integration of the gene of interest in the genome of the treated cells. This method is efficient and applicable to the cell line (e.g., HeLa cells) and primary cells (e.g., human primary keratinocytes), and thus represents a valuable tool for genetic engineering and therapeutic gene transfer.

Using the procedure presented here, three IDLV vectors carrying the regulated SB components (SB100X, rtTA2S-M2 and T2-GFP transposon) were packaged and used to efficiently deliver and tightly regulate the SB system in human cells. Figure 1A shows a scheme for in vitro transduction of HeLa cells, which was performed to evaluate the transcriptional regulation of SB transposase with the best dose combination of the two IDLV vectors (reported ...

Watch this Scientific Journal Video about An Efficient In Vitro Transposition Method by a Transcriptionally Regulated Sleeping Beauty System Packaged into an Integration Defective Lentiviral Vector at

An Efficient In Vitro Transposition Method by a Transcriptionally Regulated Sleeping Beauty System Packaged into an Integration Defective Lentiviral Vector

This protocol describes a method to achieve stable integration of a gene of interest into the human genome by the transcriptionally regulated Sleeping Beauty system. Preparation of the integration of defective lentiviral vectors, the in vitro transduction of human cells, and the molecular assay on transduced cells are reported.

An Efficient In Vitro Transposition Method by a Transcriptionally Regulated Sleeping Beauty System Packaged into an Integration Defective Lentiviral Vector

The Sleeping Beauty (SB) transposon is a non-viral integrating system with proven efficacy for gene transfer and functional genomics. To optimize the SB ...

The overall goal of this method is to achieve stable integration of a gene of interest into the human genome of primary cells by the transcriptionally regulated Sleeping Beauty system packaged into integration defective lentiviral vectors. This method can help answer key questions in gene therapy field such as integration of the gene of interest into primary cells using Sleeping Beauty system. The major advantages of this technique is that the inducible hyperactive Sleeping Beauty system is efficiently delivering primary cells resistant to several transfection methods.Demonstrating the procedure will be Daniela Benati, a post-doc in my lab. Prior to the day of transduction, the human primary keratinocytes are grown as described in the text protocol. To detach subconfluent keratinocytes in culture, remove the medium, wash the cells with 1X PBS, and add one milliliter pre-warmed trypsin working solution to each well.Incubate at 37 degrees Celsius for 15 minutes in the cell culture incubator. Resuspend the cells of a well thoroughly and transfer the cell suspension to a centrifuge tube containing two milliliters of serum-containing medium. Rinse the well once with three milliliters of 1X PBS and add the rinsed solution to the centrifuge tube with the cell suspension.Centrifuge at 580 times g for five minutes and discard the supernatant. Wash the cells with five milliliters of 1X PBS, centrifuge at 580 times g for five minutes, and discard the supernatant. In a 15 milliliter polypropylene tube, dilute 1.6 times 10 to the fifth keratinocytes in one milliliter of CFAD medium.Prepare one dilution for each condition. The three Integration Defective Lentiviral or IDLV vectors were prepared earlier as described in the text protocol. IDLVTKSB carries the TET-regulated hyperactive SB100X transposes.IDLVrtTA codes for the modulator and IDLVT2 carries the gene of interest. GFP in this example flanked by transposon inverted repeats. Dilute the lentiviral vectors in one milliliter of CFAD medium in the presence of 20 microliters of polybrene as indicated in this table.One, two, and three are negative controls without vectors. Four and five contain IDLVTKSB and IDLVrtTA. Six, seven, eight, and nine contain all three lentiviral vectors.Transduce cells in suspension by adding each lentiviral vector dilution to the corresponding keratinocytes suspension. Remove the medium from lethally irradiated 3T3-J2 feeder cells that receded a day ago and plate the transduced keratinocytes onto them. After transducing a well, proceed with the next one.Incubate at 25 degrees for 30 minutes. Then transfer the cells to 37 degrees for six hours. After six hours, add one milliliter of CFAD medium to each well.Add one micromolar of doxycycline to wells five, eight, and nine. Return cells to 37 degrees Celsius. Subsequently, process the cells in each well as described in the text protocol.For cytofluorimetric analysis, the transduced primary keratinocytes detached at day three and day six must be discriminated from the 3T3-J2 feeder layer by labeling the cells with mouse monoclonal APC conjugated antifeeder antibody. Prepare four milliliters of staining buffer for each sample by combining 5%FBS, 500 millimolar EDTA, and 1X PBS. Wash the detached cells with one milliliter of staining buffer.Centrifuge at 580 times g for five minutes and discard the supernatant. In a tube for flow cytometry acquisition, resuspend five times 10 to the fourth cells in 100 microliters of staining buffer. Add two microliters of antifeeder antibody at a dilution of one to 50 and mix well.Incubate on ice in the dark for 30 minutes. After 30 minutes, wash with two milliliters of staining buffer. Centrifuge at 580 times g for five minutes.Discard the supernatant and resuspend in 200 microliters of staining buffer. Use a flow cytometer configured with a blue laser, a red laser, and filters for detection of GFP and APC fluorescence to acquire APC and GFP signals. The GFP positive cell fraction of the APC negative cell population indicates transduced keratinocytes.To perform real-time PCR, set up reactions in triplicate in a 96-well plate in a final reaction volume of 25 microliters. Each reaction contains one microliter of cDNA, 12.5 microliters of 2X master mix, 1.25 microliters of primers in 20X probe mix, and 10.25 microliters of sterile water. Run real-time PCR according to this PCR program, one cycle at 95 degrees Celsius for 10 minutes then 40 cycles of 95 degrees Celsius for 15 seconds and 60 degrees Celsius for one minute and hold at four degrees Celsius.Lastly, analyze the real-time PCR data as described in the text protocol. Expression analysis of HeLa cells transduced with increasing doses of transposes in modulator vectors plus or minus doxycycline showed that high doses of both IDLVs were required to strongly induce SB100X expression. Quantitative analysis of SB100X expression on HeLa cells transduced with transposes vector in the presence or absence of the modulator vector revealed an eight-fold activation over the background in the presence of doxycycline.The transcriptionally regulated Sleeping Beauty system can also be used in primary cells. SB100X expression on keratinocytes transduced with transposes and modulator vectors showed a 15-fold activation over the off state. The transposition experiment performed in keratinocytes transduced with the three IDLV vectors plus or minus doxycycline indicated that 12%of cells treated with doxycycline stably integrated at least one copy of GFP in their genome.Once mastered, the vital transduction in primary cells can be done in eight hours, cytofluorimetric analysis in two hours, and expression analysis in three hours if performed properly. While attempting this procedure, it's important to remember to optimize vector dose combination used to transduce primary cells and to achieve the higher expression level of the inducible Sleeping Beauty transposes. Following this procedure, other methods such as quantitative PCR and linker-mediated PCR can be performed in order to answer additional questions such as transposon copy number per cell and integration for filing the genome.After its development, this technique paved the way for the researcher in the field of gene therapy to explore the application of Sleeping Beauty system in engineering clinical relevant cells. After watching the video, you should have good understanding of how to first transduce primary keratinocytes with the IDLV vectors. Second, measure the expression of hyperactive transposes upon doxycycline condition and finally follow transposition by flow cytometry.Don't forget that working with viral vectors can be extremely hazardous so precautions such as BCL to contain the level and the disposable supply should always be taken with you while performing these procedures.

Research

JoVE Journal

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