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.

<ol>
	<li>Mates, L., 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=Molecular+evolution+of+a+novel+hyperactive+Sleeping+Beauty+transposase+enables+robust+stable+gene+transfer+in+vertebrates.">Molecular evolution of a novel hyperactive Sleeping Beauty transposase enables robust stable gene transfer in vertebrates.</a> Nat Genet. 41 (6), 753-761 (2009).</li><li>Maiti, S. 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. A., 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=Derivation+and+characterization+of+sleeping+beauty+transposon-mediated+porcine+induced+pluripotent+stem+cells.">Derivation and characterization of sleeping beauty transposon-mediated porcine induced pluripotent stem cells.</a> Stem Cells Dev. 22 (1), 124-135 (2013).</li><li>Turchiano, G., 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=Genomic+analysis+of+Sleeping+Beauty+transposon+integration+in+human+somatic+cells.">Genomic analysis of Sleeping Beauty transposon integration in human somatic cells.</a> PLoS One. 9 (11), e112712 (2014).</li><li>Jin, Z., 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=The+hyperactive+Sleeping+Beauty+transposase+SB100X+improves+the+genetic+modification+of+T+cells+to+express+a+chimeric+antigen+receptor.">The hyperactive Sleeping Beauty transposase SB100X improves the genetic modification of T cells to express a chimeric antigen receptor.</a> Gene Ther. 18 (9), 849-856 (2011).</li><li>Latella, M. 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. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mobilization+of+DNA+transposable+elements+from+lentiviral+vectors.">Mobilization of DNA transposable elements from lentiviral vectors.</a> Mob Genet Elements. 1 (2), 139-144 (2011).</li><li>Green, M. R., Sambrook, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular Cloning: A laboratory Manual. , (2012).</li><li>Dellambra, 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=Corrective+transduction+of+human+epidermal+stem+cells+in+laminin-5-dependent+junctional+epidermolysis+bullosa.">Corrective transduction of human epidermal stem cells in laminin-5-dependent junctional epidermolysis bullosa.</a> Hum Gene Ther. 9, 1359-1370 (1998).</li></ol>

The authors have nothing to disclose.

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 years10,12,15. However, none of the viral vectors employed up to now addressed the issue of constitutive expression of transposase that could result in the risk of uncontrollable rehopping of the transposon.
To transcriptionally regulate transposase expression, we took advantage of a modified Tet-ON system16. The minimal TK promoter fused to the TetO element, upon binding to the rtTA2s-M2 modulator in the presence of dox, conferred a fine-tuning of transposase activation over the background (off-state, in the absence of dox). The regulatory performance depends on the appropriate IDLVs (IDLVTKSB and IDLVrtTA2s-M2) vector dose combination and on cell types (e.g., HeLa cells and human primary keratinocytes). Independently from target cells, the vector dose must ensure the highest level of transduction of the target cells without affecting cell viability, thus an escalating dose-combination experiment in HeLa cells (or other cell lines) to define the optimal doses, by semi-quantitative RT-PCR, is strongly suggested.
Transposition of the GOI was assessed in primary cells. Human primary keratinocytes seeded on lethally irradiated 3T3-J2 feeder layer were transduced with 3 IDLVs (IDLVTKSB, IDLVrtTA2s-M2 and IDLVT2) at the experimentally defined doses. A clear dox-dependent activation of the transposase was demonstrated by qRT-PCR. Moreover, stable integration of the GOI was demonstrated by cytofluorimetric analysis of GFP expression in cells transduced in the presence or absence of dox, indicating the possibility to employ the IDLV platform to vehicle the transcriptionally regulated SB components to the primary cells, and the successful integration of a GOI into the genome of target cells.
The critical steps in the protocol are IDLV vector production and transduction of target cells at the optimal efficiency and vector dose combination. Low transfection efficiency of HEK293T cells may result in low vector yield. The definition of doses of IDLVTKSB and IDLVrtTA2s-M2 is necessary to avoid an unbalance of Tet-ON components that may result in high leakiness in the absence of the modulator or in low fold induction of the TetOTK promoter upon dox treatment. These critical steps could be addressed by minor modifications. For instance, the transfection protocol could be standardized using commercial transfection reagents. The transduction protocol should be adapted according to cell type of interest, particularly considering whether spinoculation increases transduction efficiency without affecting cell viability. Vectors and polybrene could be added to the culture medium of the target cells and replaced with fresh medium after 6 h or overnight incubation. It is also worth noting that the percentage of GFP+ cells 5 days after transduction could be affected by the background integration of IDLVs.
A minor limitation of this method is that the IDLV vectors are produced transiently by transfection of HEK293T cells. Depending on the vector doses to be used, repeated vector productions are required. A second limitation is related to the length of the GOI, as the cargo capacity of the IDLV vector is approximately 8 kb.
In conclusion, this method here allows the integration of a GOI through transcriptionally regulated SB components packaged into IDLV vectors, which, among the existing methods to deliver SB system, show a broad range of tropism and high transduction efficiency. Moreover, the modified Tet-ON system permits a transcriptional regulation of the SB100X expression, a relevant issue in the risk of transposon re-hopping or in multiple serial transposition events if they are required. Possible applications of this method include genome engineering of cell lines (e.g., HeLa cells, HEK293 cells) to establish stable packaging cells for viral vectors, and integration of therapeutic genes into clinical relevant primary cells.

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 in Table 2). The transcriptional regulation of SB100X was measured by qRT-PCR (Figure 1B) and plotted as a relative quantity (RQ) value with respect to the off-state (IDLVTKSB alone RQ=1). In transduced HeLa cells, a 8-fold activation (+dox) of SB100X expression over the background (IDLVTKSB) was obtained. Notably, cotransduced HeLa cells in the absence of dox showed transposase expression comparable to the off states, indicating a tight control of TetTK promoter by rtTA2s-M2 modulator.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56742/56742fig1.jpg" /> 
Figure 1: Transduction of HeLa cells for the expression of transcriptionally-regulated SB transposase carried by IDLV vectors. (A) A scheme for in vitro transduction of HeLa cells with IDLVs for the expression of transposase. (B) qRT-PCR analysis of HeLa cells transduced with IDLVTKSB (2,600 ng of p24) in the presence (+) or absence (-) of IDLVrtTA2S-M2 (9,600 ng of p24) and 1 &#181;M doxycycline (dox). The dashed line connects samples showing significantly different values (***P &#60; 0.005). The experiment was performed in triplicate. Mean S.E.M is shown as error bars. (Figure modified from Cocchiarella, F. et al., 201618). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56742/56742fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
As an example, Figure 2 shows the dose ranging experiment set up to reach the optimal transcriptional regulation of transposase. HeLa cells were transduced with increasing doses (130, 260 , and 2,600 ng of p24) of IDLVTKSB in combination with IDLVrtTA2s-M2 (960 and 9,600 ng of p24), in the presence or absence of dox. Semi-quantitative RT-PCR analysis clearly shows that high doses of both IDLVs were required to strongly induce SB100X expression. Different target cell type and variations in viral vector titration may result in sup-optimal SB expression, and we therefore recommend performing dosage setting experiments.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56742/56742fig2.jpg" /> 
Figure 2: IDLV dose setting experiment in HeLa cells to define the optimal dose combination of the two IDLVs. Semi-quantitative RT-PCR analysis on HeLa cells co-transduced with different doses of IDLVTKSB vector (130, 260, and 2,600 ng of p24) in the absence or presence of IDLVrtTA2s-M2 vector (960 and 9,600 ng of p24) and dox. The GAPDH was amplified as a standard control. SB100X transposase and rtTA2s-M2 expression are reported in all tested conditions; NC = negative control. (Figure modified from Cocchiarella, F. et al., 201618). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56742/56742fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The transcriptional regulated SB system could also be used in primary cells, particularly in those cells resistant to several transfection methods, such as human primary keratinocytes. Figure 3 shows a scheme for transduction of primary keratinocytes cultivated on lethally irradiated 3T3-J2 feeder layer, with IDLV vectors carrying the regulated SB components, in the presence or absence of dox to induce SB100X expression.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56742/56742fig3.jpg" /> 
Figure 3: Transduction in suspension of human primary keratinocytes with IDLV vectors for the expression of transcriptionally-regulated SB transposase and transposon. A scheme for in vitro transduction of human primary keratinocytes with IDLVs to express transcriptionally-regulated SB transposase and transposon, in the presence or absence of 1 &#181;M dox. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56742/56742fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The qRT-PCR analysis in Figure 4A shows that the best dose combination of transposase and modulator vectors, reported in Table 2, resulted in 15-fold activation of SB expression over the off-state (-dox). The transposition experiment was performed in keratinocytes transduced with the three IDLV vectors (IDLVTKSB, IDLVrtTA2s-M2 and IDLVT2) in the presence or absence of dox. Expression of the GOI (GFP) beared by T2-transposon, in the APC- compartment, was measured by flow cytometer analysis 2 days post-transduction (dpt) and at the endopoint of the culture (5 days for human primary keratinocytes). The endpoint was reached when unintegrated T2-GFP transposon dropped to a barely detectable level (an average of 0.6%) due to cell division-dependent dilution. Figure 4B shows the ratio between the percentage of GFP+ cells in the APC- compartment at the endpoint and 2 days post transduction, normalized to the residual level of the transposon alone. This indicates the percentage of cells hosting at least 1 copy of the expressed GOI stably integrated in their genome, assessed at 12% for human primary keratinocytes.
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56742/56742fig4v2.jpg" /> 
Figure 4: Analysis of the expression of transposase and GFP in transduced human primary keratinocytes. (A) qRT-PCR assay on primary keratinocytes transduced with IDLVTKSB and IDLVrtTA2S-M2 in the presence or absence of 1 &#181;M dox. (B) Co-transduction of primary keratinocytes with IDLVT2 transposon and IDLVs for transposase expression in presence or absence of dox. On Y-axis, %GFP+ cells 5dpt / %GFP+ cells 2 dpt * 100 (mean &#177; SEM of two experiments). ** show significantly different values (P &#60; 0.05). (Figure modified from Cocchiarella, F. et al., 201618). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56742/56742fig4v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Plasmid</td>
			<td>Description</td>
			<td>References</td>
		</tr>
		<tr>
			<td>pCCL-TetOTKSB</td>
			<td>Transfer plasmid used to generate IDLVTKSB vector. This plasmid derived from pTetOTKSB, after cloning TetOTKSB in the pCCL lentiviral backbone.</td>
			<td>18, 20</td>
		</tr>
		<tr>
			<td>pCCL-PGKrtTA2S-M2</td>
			<td>Transfer plasmid used to generate IDLVrtTA2s-M2 vector.</td>
			<td>provided by Prof. V. Zappavigna</td>
		</tr>
		<tr>
			<td>pCCL-T2GFP</td>
			<td>Transfer plasmid used to generate IDLVT2 vector. This plasmid derived from pT2GFP, after cloning the GFP between the T2-transposon IRs in the pCCL lentiviral backbone.</td>
			<td>18, 20</td>
		</tr>
		<tr>
			<td>pMD.Lg/pRRE.D64VInt</td>
			<td>Packaging plasmid to generate IDLV vector.</td>
			<td>19</td>
		</tr>
		<tr>
			<td>pMD2.G</td>
			<td>Plasmid coding for VSV-G envelope to generate lentiviral vector.</td>
			<td>20</td>
		</tr>
		<tr>
			<td>pRSV-Rev</td>
			<td>Rev plasmid to generate lentiviral vector.</td>
			<td>20</td>
		</tr>
		<tr>
			<td>Vector</td>
			<td colspan="2">Description</td>
		</tr>
		<tr>
			<td>IDLVTKSB</td>
			<td colspan="2">Integration defective lentiviral vector for the expression of the SB100X under the control of Tet-responsive TK promoter.</td>
		</tr>
		<tr>
			<td>IDLVrtTA2s-M2</td>
			<td colspan="2">Integration defective lentiviral vector for the expression of the rtTA2s-M2 modulator under the control of PGK promoter.</td>
		</tr>
		<tr>
			<td>IDLVT2</td>
			<td colspan="2">Integration defective lentiviral vector for the expression of the GOI cloned between the T2-transposon IRs.</td>
		</tr>
	</tbody>
</table>
Table 1: Plasmids and vectors employed in this protocol. Descriptions and references are provided.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Vectors</td>
			<td>HeLa (2x105 cells)</td>
			<td>Keratinocytes (1.6x105 cells)</td>
		</tr>
		<tr>
			<td>IDLVTKSB</td>
			<td>2,600 ng p24</td>
			<td>13,000 ng p24</td>
		</tr>
		<tr>
			<td>IDLVrtTA2s-M2</td>
			<td>9,600 ng p24</td>
			<td>48,000 ng p24</td>
		</tr>
		<tr>
			<td>IDLVT2</td>
			<td></td>
			<td>9,160 ng p24</td>
		</tr>
	</tbody>
</table>
Table 2: Optimized vector doses for transduction of HeLa cells and human primary keratinocytes.

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 ...

an-efficient-vitro-transposition-method-transcriptionally-regulated

Nanyang Technological University

Research

JoVE Journal

Biology

8.8K Views.  University of Modena and Reggio Emilia. 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.

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

Production of Replication-Defective Retrovirus by Transient Transfection of 293T cells

Our lab studies human myeloproliferative diseases induced by such oncogenes as Bcr-Abl or growth factor receptor-derived oncogenes (ZNF198-FGFR1, Bcr-PDGFR&#945;, etc.). We are able to model and study a human-like disease in our mouse model, by transplanting bone marrow cells previously infected with a retrovirus expressing the oncogene of interest. Replication-defective retrovirus encoding a human oncogene and a marker (GFP, RFP, antibiotic resistance gene, etc.) is produced by a transient transfection protocol using 293T cells, a human renal epithelial cell line transformed by the adenovirus E1A gene product. 293 cells have the unusual property of being highly transfectable by calcium phosphate (CaPO4), with up to 50-80% transfection efficiency readily attainable. Here, we co-transfect 293 cells with a retroviral vector expressing the oncogene of interest and a plasmid that expresses the gag-pol-env packaging functions, such as the single-genome packaging constructs kat or pCL, in this case the EcoPak plasmid. The initial transfection is further improved by use of chloroquine. Stocks of ecotropic virus, collected as culture supernatant 48 hrs. post-transfection, can be stored at -80&deg;C and used for infection of cell-lines in view of transformation and in vitro studies, or primary cells such as mouse bone marrow cells, that can then be used for transplant in our mouse model.

This technique demonstrates an efficient way to prepare replication-defective retroviral stocks encoding a human oncogene, and subsequently used for induction of myeloproliferative disease in the mouse model.

Our lab studies human myeloproliferative diseases induced by such oncogenes as Bcr-Abl or growth factor receptor-derived oncogenes (ZNF198-FGFR1, ...

Homemade Site Directed Mutagenesis of Whole Plasmids

Site directed mutagenesis of whole plasmids is a simple way to create slightly different variations of an original plasmid. With this method the cloned target gene can be altered by substitution, deletion or insertion of a few bases directly into a plasmid. It works by simply amplifying the whole plasmid, in a non PCR-based thermocycling reaction. During the reaction mutagenic primers, carrying the desired mutation, are integrated into the newly synthesized plasmid. In this video tutorial we demonstrate an easy and cost effective way to introduce base substitutions into a plasmid. The protocol works with standard reagents and is independent from commercial kits, which often are very expensive. Applying this protocol can reduce the total cost of a reaction to an eighth of what it costs using some of the commercial kits. In this video we also comment on critical steps during the process and give detailed instructions on how to design the mutagenic primers.

Site directed mutagenesis of whole plasmids is a simple way to create slightly different variations of an original plasmid. Here we demonstrate an easy and cost effective way to introduce base substitutions into a plasmid using standard reagents.

Site directed mutagenesis of whole plasmids is a simple way to create slightly different variations of an original plasmid. With this method the cloned ...

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells (iPSCs) Using Retroviral Vector with GFP

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has been previously shown that human somatic cells can be reprogrammed to pluripotency by ectopic expression of four transcription factors (Oct4, Sox2, Klf4 and Myc) and become induced pluripotent stem cells (iPSCs)2-4 . Like hESCs, human iPSCs are pluripotent and a potential source for autologous cells. Here we describe the protocol to reprogram human fibroblast cells with the four reprogramming factors cloned into GFP-containing retroviral backbone4. Using the following protocol, we generate human iPSCs in 3-4 weeks under human ESC culture condition. Human iPSC colonies closely resemble hESCs in morphology and display the loss of GFP fluorescence as a result of retroviral transgene silencing. iPSC colonies isolated mechanically under a fluorescence microscope behave in a similar fashion as hESCs. In these cells, we detect the expression of multiple pluripotency genes and surface markers.

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells iPSCs Using Retroviral Vector with GFP

A method to generate human induced pluripotent stem cells (iPSCs) via retrovirus-mediated ectopic expression of OCT4, SOX2, KLF4 and MYC is described. A practical way to identify human iPSC colonies based on GFP expression is also discussed.

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has ...

A Step-by-step Method for the Reconstitution of an ABC Transporter into Nanodisc Lipid Particles

The nanodisc is a discoidal particle (~ 10-12 nm large) that trap membrane proteins into a small patch of phospholipid bilayer. The nanodisc is a particularly attractive option for studying membrane proteins, especially in the context of ligand-receptor interactions. The method pioneered by Sligar and colleagues is based on the amphipathic properties of an engineered highly a-helical scaffold protein derived from the apolipoprotein A1. The hydrophobic faces of the scaffold protein interact with the fatty acyl side-chains of the lipid bilayer whereas the polar regions face the aqueous environment. Analyses of membrane proteins in nanodiscs have significant advantages over liposome because the particles are small, homogeneous and water-soluble. In addition, biochemical and biophysical methods normally reserved to soluble proteins can be applied, and from either side of the membrane. In this visual protocol, we present a step-by-step reconstitution of a well characterized bacterial ABC transporter, the MalE-MalFGK2 complex. The formation of the disc is a self-assembly process that depends on hydrophobic interactions taking place during the progressive removal of the detergent. We describe the essential steps and we highlight the importance of choosing a correct protein-to-lipid ratio in order to limit the formation of aggregates and larger polydisperse liposome-like particles. Simple quality controls such as gel filtration chromatography, native gel electrophoresis and dynamic light scattering spectroscopy ensure that the discs have been properly reconstituted.

Nanodiscs are small discoid particles that incorporate membrane proteins into a small patch of phospholipid bilayer. We provide a visual protocol that shows the step-by-step incorporation of the MalFGK2 transporter into a disc.

The nanodisc is a discoidal particle (~ 10-12 nm large) that trap membrane proteins into a small patch of phospholipid bilayer. The nanodisc is a ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

Hybrid-Cut: An Improved Sectioning Method for Recalcitrant Plant Tissue Samples

Maintaining plant section integrity is essential for studying detailed anatomical structures at the cellular, tissue, or even organ level. However, some plant cells have rigid cell walls, tough fibers and crystals (calcium oxalate, silica, etc.), and high water content that often disrupt tissue integrity during plant tissue sectioning.
This study establishes a simple Hybrid-Cut tissue sectioning method. This protocol modifies a paraffin-based sectioning technique and improves the integrity of tissue sections from different plants. Plant tissues were embedded in paraffin before sectioning in a cryostat at -16 &#176;C. Sectioning under low temperature hardened the paraffin blocks, reduced tearing and scratching, and improved tissue integrity significantly. This protocol was successfully applied to calcium oxalate-rich Phalaenopsis orchid tissues as well as recalcitrant tissues such as reproductive organs and leaves of rice, maize, and wheat. In addition, the high quality of tissue sections from Hybrid-Cut could be used in combination with in situ hybridization (ISH) to provide spatial expression patterns of genes of interest. In conclusion, this protocol is particularly useful for recalcitrant plant tissue containing high crystal or silica content. Good quality tissue sections enable morphological and other biological studies.

This protocol describes a simple Hybrid-Cut tissue sectioning method that is useful for recalcitrant plant tissues. Good quality tissue sections enable anatomical studies and other biological studies including in situ hybridization (ISH).

Maintaining plant section integrity is essential for studying detailed anatomical structures at the cellular, tissue, or even organ level. However, some ...

An In Vitro Model for Studying Cellular Transformation by Kaposi Sarcoma Herpesvirus

Kaposi sarcoma (KS) is an unusual tumor composed of proliferating spindle cells that is initiated by infection of endothelial cells (EC) with KSHV, and develops most often in the setting of immunosuppression. Despite decades of research, optimal treatment of KS remains poorly defined and clinical outcomes are especially unfavorable in resource-limited settings. KS lesions are driven by pathological angiogenesis, chronic inflammation, and oncogenesis, and various in vitro cell culture models have been developed to study these processes. KS arises from KSHV-infected cells of endothelial origin, so EC-lineage cells provide the most appropriate in vitro surrogates of the spindle cell precursor. However, because EC have a limited in vitro lifespan, and as the oncogenic mechanisms employed by KSHV are less efficient than those of other tumorigenic viruses, it has been difficult to assess the processes of transformation in primary or telomerase-immortalized EC. Therefore, a novel EC-based culture model was developed that readily supports transformation following infection with KSHV. Ectopic expression of the E6 and E7 genes of human papillomavirus type 16 allows for extended culture of age- and passage-matched mock- and KSHV-infected EC and supports the development of a truly transformed (i.e., tumorigenic) phenotype in infected cell cultures. This tractable and highly reproducible model of KS has facilitated the discovery of several essential signaling pathways with high potential for translation into clinical settings.

An In Vitro Model for Studying Cellular Transformation by Kaposi Sarcoma Herpesvirus

Kaposi sarcoma (KS) is a tumor induced by infection with the oncogenic virus human herpesvirus-8/KS herpesvirus (HHV-8/KSHV). The endothelial cell culture model described here is uniquely suited for studying the mechanisms by which KSHV transforms host cells.

Kaposi sarcoma (KS) is an unusual tumor composed of proliferating spindle cells that is initiated by infection of endothelial cells (EC) with KSHV, and ...

An Efficient and Flexible Cell Aggregation Method for 3D Spheroid Production

Monolayer cell culture does not adequately model the in vivo behavior of tissues, which involves complex cell-cell and cell-matrix interactions. Three-dimensional (3D) cell culture techniques are a recent innovation developed to address the shortcomings of adherent cell culture. While several techniques for generating tissue analogues in vitro have been developed, these methods are frequently complex, expensive to establish, require specialized equipment, and are generally limited by compatibility with only certain cell types. Here, we describe a rapid and flexible protocol for aggregating cells into multicellular 3D spheroids of consistent size that is compatible with growth of a variety of tumor and normal cell lines. We utilize varying concentrations of serum and methyl cellulose (MC) to promote anchorage-independent spheroid generation and prevent the formation of cell monolayers in a highly reproducible manner. Optimal conditions for individual cell lines can be achieved by adjusting MC or serum concentrations in the spheroid formation medium. The 3D spheroids generated can be collected for use in a wide range of applications, including cell signaling or gene expression studies, candidate drug screening, or in the study of cellular processes such as tumor cell invasion and migration. The protocol is also readily adapted to generate clonal spheroids from single cells, and can be adapted to assess anchorage-independent growth and anoikis-resistance. Overall, our protocol provides an easily modifiable method for generating and utilizing 3D cell spheroids in order to recapitulate the 3D microenvironment of tissues and model the in vivo growth of normal and tumor cells.

Here, we describe a rapid and flexible protocol for the formation of 3D cell spheroids through cell aggregation. This is easily adapted to multiple cell types and is suitable for use in a variety of applications including cell migration, invasion, or anoikis assays, and for imaging and quantifying cell-matrix interactions.

Monolayer cell culture does not adequately model the in vivo behavior of tissues, which involves complex cell-cell and cell-matrix interactions. ...

IR-TEx: An Open Source Data Integration Tool for Big Data Transcriptomics Designed for the Malaria Vector Anopheles gambiae

IR-TEx is an application written in Shiny (an R package) that allows exploration of the expression of (as well as assigning functions to) transcripts whose expression is associated with insecticide resistance phenotypes in Anopheles gambiae mosquitoes. The application can be used online or downloaded and used locally by anyone. The local application can be modified to add new insecticide resistance datasets generated from multiple -omics platforms. This guide demonstrates how to add new datasets and handle missing data. Furthermore, IR-TEx can be completely and easily recoded to use-omics datasets from any experimental data, making it a valuable resource to many researchers. The protocol illustrates the utility of IR-TEx in identifying new insecticide resistance candidates using the the microsomal glutathione transferase, GSTMS1, as an example. This transcript is upregulated in multiple pyrethroid resistant populations from C&#244;te D&#39;Ivoire and Burkina Faso. The identification of co-correlated transcripts provides further insight into the putative roles of this gene.

IR-TEx: An Open Source Data Integration Tool for Big Data Transcriptomics Designed for the Malaria Vector Anopheles gambiae

IR-TEx explores insecticide resistance-related transcriptional profiles in the species Anopheles gambiae. Provided here are full instructions for using the application, modifications for exploring multiple transcriptomic datasets, and using the framework to build an interactive database for collections of transcriptomic data from any organism, generated in any platform.

IR-TEx is an application written in Shiny (an R package) that allows exploration of the expression of (as well as assigning functions to) transcripts ...

Imaging Molecular Adhesion in Cell Rolling by Adhesion Footprint Assay

Rolling adhesion, facilitated by selectin-mediated interactions, is a highly dynamic, passive motility in recruiting leukocytes to the site of inflammation. This phenomenon occurs in postcapillary venules, where blood flow pushes leukocytes in a rolling motion on the endothelial cells. Stable rolling requires a delicate balance between adhesion bond formation and their mechanically-driven dissociation, allowing the cell to remain attached to the surface while rolling in the direction of flow. Unlike other adhesion processes occurring in relatively static environments, rolling adhesion is highly dynamic as the rolling cells travel over thousands of microns at tens of microns per second. Consequently, conventional mechanobiology methods such as traction force microscopy are unsuitable for measuring the individual adhesion events and the associated molecular forces due to the short timescale and high sensitivity required. Here, we describe our latest implementation of the adhesion footprint assay to image the P-selectin: PSGL-1 interactions in rolling adhesion at the molecular level. This method utilizes irreversible DNA-based tension gauge tethers to produce a permanent history of molecular adhesion events in the form of fluorescence tracks. These tracks can be imaged in two ways: (1) stitching together thousands of diffraction-limited images to produce a large field of view, enabling the extraction of adhesion footprint of each rolling cell over thousands of microns in length, (2) performing DNA-PAINT to reconstruct super-resolution images of the fluorescence tracks within a small field of view. In this study, the adhesion footprint assay was used to study HL-60 cells rolling at different shear stresses. In doing so, we were able to image the spatial distribution of the P-selectin: PSGL-1 interaction and gain insight into their molecular forces through fluorescence intensity. Thus, this method provides the groundwork for the quantitative investigation of the various cell-surface interactions involved in rolling adhesion at the molecular level.

This protocol presents the experimental procedures to perform the adhesion footprint assay to image the adhesion events during fast cell rolling adhesion.