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&amp;rsquo;s modified Eagle&amp;rsquo;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 (Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;&amp;middot;2H&lt;sub&gt;2&lt;/sub&gt;O), 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 &amp;micro;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&amp;rsquo;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>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 &amp;deg;C, -20 &amp;deg;C, -80 &amp;deg;C), micropipettes and pipet-aid).</td><td></td><td></td><td></td></tr><tr><td>Protective laboratory coats, gloves, face protection in accordance with Institute rules and regulations, in particular for lentiviral particle production and transduction.</td><td></td><td></td><td></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

8.9K Views.  University of Modena and Reggio Emilia. Dit protocol beschrijft een methode om stabiele integratie van een gen van belang in het menselijk genoom door het transcriptionally gereglementeerde Sleeping Beauty -systeem. Voorbereiding van de integratie van defecte lentivirale vectoren, de in vitro -transductie van menselijke cellen en de moleculaire test op getransduceerde cellen worden gemeld.

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

A Protocol for the Production of Integrase-deficient Lentiviral Vectors for CRISPR/Cas9-mediated Gene Knockout in Dividing Cells

Lentiviral vectors are an ideal choice for delivering gene-editing components to cells due to their capacity for stably transducing a broad range of cells and mediating high levels of gene expression. However, their ability to integrate into the host cell genome enhances the risk of insertional mutagenicity and thus raises safety concerns and limits their usage in clinical settings. Further, the persistent expression of gene-editing components delivered by these integration-competent lentiviral vectors (ICLVs) increases the probability of promiscuous gene targeting. As an alternative, a new generation of integrase-deficient lentiviral vectors (IDLVs) has been developed that addresses many of these concerns. Here the production protocol of a new and improved IDLV platform for CRISPR-mediated gene editing and list the steps involved in the purification and concentration of such vectors is described and their transduction and gene-editing efficiency using HEK-293T cells was demonstrated. This protocol is easily scalable and can be used to generate high titer IDLVs that are capable of transducing cells in vitro and in vivo. Moreover, this protocol can be easily adapted for the production of ICLVs.

We describe the production strategy of integrase-deficient lentiviral vectors (IDLVs) as vehicles for delivering CRISPR/Cas9 to cells. With an ability to mediate quick and robust gene editing in cells, IDLVs present a safer and equally effective vector platform for gene delivery compared to integrase-competent vectors.

Lentiviral vectors are an ideal choice for delivering gene-editing components to cells due to their capacity for stably transducing a broad range of cells ...

Genetics

Packaging HIV- or FIV-based Lentivector Expression Constructs &amp; Transduction of VSV-G Pseudotyped Viral Particles

As with standard plasmid vectors, it is possible to transfect lentivectors in plasmid form into cells with low-to-medium efficiency to obtain transient expression of effectors. Packaging lentiviral expression constructs into pseudoviral particles, however, enables up to 100% transduction, even with difficult-to-transfect cells, such as primary, stem, and differentiated cells. Moreover, the lentiviral delivery does not produce the specific cellular responses typically associated with chemical transfections, such as cell death resulting from toxicity of the transfection reagent 1, 2. When transduced into target cells, the lentiviral construct integrates into genomic DNA and provides stable expression of the small hairpin RNA (shRNA), cDNA, microRNA or reporter gene 3, 4. Target cells stably expressing the effector molecule can be isolated using a selectable marker contained in the expression vector construct such as puromycin or GFP. After pseudoviral particles infect target cells, they cannot replicate within target cells because the viral structural genes are absent and the long terminal repeats (LTRs) are designed to be self-inactivating upon transduction 5, 6.
There are three main components necessary for efficient lentiviral packaging 1, 5, 6, 7. 
1. The lentiviral expression vector that contains some of the genetic elements required for packaging, stable integration of the viral expression construct into genomic DNA, and expression of the effector or reporter. 
2. The lentiviral packaging plasmids that provide the proteins essential for transcription and packaging of an RNA copy of the expression construct into recombinant pseudoviral particles. This protocol uses the pPACK plasmids (SBI) that encode for gag, pol, and rev from the HIV or FIV genome and Vesicular Stomatitis Virus g protein (VSV-G) for the viral coat protein. 
3. 293TN producer cells (derived from HEK293 cells) that express the SV40 large T antigen, which is required for high-titer lentiviral production and a neomycin resistance gene, useful for reselecting the cells for maintenance.
An overview of the viral production protocol can be seen in Figure 1. Viral production starts by co-transfecting 293TN producer cells with the lentiviral expression vector and the packaging plasmids. Viral particles are secreted into the media. After 48-72 hours the cell culture media is harvested. Cellular debris is removed from the cell culture media, and the viral particles are precipitated by centrifugation with PEG-it for concentration. Produced lentiviral particles are then titered and can be used to transduce target cells. Details of viral titering are not included in this protocol, but can be found at:
<a href="http://www.systembio.com/downloads/global_titer_kit_web_090710.pdf" target="_blank">http://www.systembio.com/downloads/global_titer_kit_web_090710.pdf</a> 8.
This protocol has been optimized using the specific products indicated. Other reagents may be substituted, but the same results cannot be guaranteed.

Packaging HIV- or FIV-based Lentivector Expression Constructs & Transduction of VSV-G Pseudotyped Viral Particles

Lentiviral expression vectors are the most effective vehicles for stably expressing different effector molecules or reporter constructs in dividing and non-dividing mammalian cells and whole organisms. Here we provide a protocol on how to package lentivector expression constructs in pseudoviral particles and to transduce target cells using the pseudoviral particles.

As with standard plasmid vectors, it is possible to transfect lentivectors in plasmid form into cells with low-to-medium efficiency to obtain transient ...

Immunology and Infection

Lentiviral Mediated Production of Transgenic Mice: A Simple and Highly Efficient Method for Direct Study of Founders

For almost 40 years, pronuclear DNA injection represents the standard method to generate transgenic mice with random integration of transgenes. Such a routine procedure is widely utilized throughout the world and its main limitation resides in the poor efficacy of transgene integration, resulting in a low yield of founder animals. Only few percent of animals born after implantation of injected fertilized oocytes have integrated the transgene. In contrast, lentiviral vectors are powerful tools for integrative gene transfer and their use to transduce fertilized oocytes allows highly efficient production of founder transgenic mice with an average yield above 70%. Furthermore, any mouse strain can be used to produce transgenic animal and the penetrance of transgene expression is extremely high, above 80% with lentiviral mediated transgenesis compared to DNA microinjection. The size of the DNA fragment that can be cargo by the lentiviral vector is restricted to 10 kb and represents the major limitation of this method. Using a simple and easy to perform injection procedure beneath the zona pellucida of fertilized oocytes, more than 50 founder animals can be produced in a single session of microinjection. Such a method is highly adapted to perform, directly in founder animals, rapid gain and loss of function studies or to screen genomic DNA regions for their ability to control and regulate gene expression in vivo.

Here, we present a protocol to promote transgene integration and production of founder transgenic mice with high efficacy by a simple injection of a lentiviral vector in the perivitelline space of a fertilized oocyte.

For almost 40 years, pronuclear DNA injection represents the standard method to generate transgenic mice with random integration of transgenes. Such a ...

Lentiviral Vector Platform for the Efficient Delivery of Epigenome-editing Tools into Human Induced Pluripotent Stem Cell-derived Disease Models

The use of hiPSC-derived cells represents a valuable approach to study human neurodegenerative diseases. Here, we describe an optimized protocol for the differentiation of hiPSCs derived from a patient with the triplication of the alpha-synuclein gene (SNCA) locus into Parkinson&#8217;s disease (PD)-relevant dopaminergic neuronal populations. Accumulating evidence has shown that high levels of SNCA are causative for the development of PD. Recognizing the unmet need to establish novel therapeutic approaches for PD, especially those targeting the regulation of SNCA expression, we recently developed a CRISPR/dCas9-DNA-methylation-based system to epigenetically modulate SNCA transcription by enriching methylation levels at the SNCA intron 1 regulatory region. To deliver the system, consisting of a dead (deactivated) version of Cas9 (dCas9) fused with the catalytic domain of the DNA methyltransferase enzyme 3A (DNMT3A), a lentiviral vector is used. This system is applied to cells with the triplication of the SNCA locus and reduces the SNCA-mRNA and protein levels by about 30% through the targeted DNA methylation of SNCA intron 1. The fine-tuned downregulation of the SNCA levels rescues disease-related cellular phenotypes. In the current protocol, we aim to describe a step-by-step procedure for differentiating hiPSCs into neural progenitor cells (NPCs) and the establishment and validation of pyrosequencing assays for the evaluation of the methylation profile in the SNCA intron 1. To outline in more detail the lentivirus-CRISPR/dCas9 system used in these experiments, this protocol describes how to produce, purify, and concentrate lentiviral vectors and to highlight their suitability for epigenome- and genome-editing applications using hiPSCs and NPCs. The protocol is easily adaptable and can be used to produce high titer lentiviruses for in vitro and in vivo applications.

Targeted DNA epigenome editing represents a powerful therapeutic approach. This protocol describes the production, purification, and concentration of all-in-one lentiviral vectors harboring the CRISPR-dCas9-DNMT3A transgene for epigenome-editing applications in human induced pluripotent stem cell (hiPSC)-derived neurons.

The use of hiPSC-derived cells represents a valuable approach to study human neurodegenerative diseases. Here, we describe an optimized protocol for the ...

Production of Lentiviral Vectors for Transducing Cells from the Central Nervous System

Efficient gene delivery in the central nervous system (CNS) is important in studying gene functions, modeling neurological diseases and developing therapeutic approaches. Lentiviral vectors are attractive tools in transduction of neurons and other cell types in CNS as they transduce both dividing and non-dividing cells, support sustained expression of transgenes, and have relatively large packaging capacity and low toxicity 1-3. Lentiviral vectors have been successfully used in transducing many neural cell types in vitro 4-6 and in animals 7-10.
Great efforts have been made to develop lentiviral vectors with improved biosafety and efficiency for gene delivery. The current third generation replication-defective and self-inactivating (SIN) lentiviral vectors are depicted in Figure 1. The required elements for vector packaging are split into four plasmids. In the lentiviral transfer plasmid, the U3 region in the 5' long terminal repeat (LTR) is replaced with a strong promoter from another virus. This modification allows the transcription of the vector sequence independent of HIV-1 Tat protein that is normally required for HIV gene expression 11. The packaging signal (&Psi;) is essential for encapsidation and the Rev-responsive element (RRE) is required for producing high titer vectors. The central polypurine tract (cPPT) is important for nuclear import of the vector DNA, a feature required for transducing non-dividing cells 12. In the 3' LTR, the cis-regulatory sequences are completely removed from the U3 region. This deletion is copied to 5' LTR after reverse transcription, resulting in transcriptional inactivation of both LTRs. Plasmid pMDLg/pRRE contains HIV-1 gag/pol genes, which provide structural proteins and reverse transcriptase. pRSV-Rev encodes Rev which binds to the RRE for efficient RNA export from the nucleus. pCMV-G encodes the vesicular stomatitis virus glycoprotein (VSV-G) that replaces HIV-1 Env. VSV-G expands the tropism of the vectors and allows concentration via ultracentrifugation 13. All the genes encoding the accessory proteins, including Vif, Vpr, Vpu, and Nef are excluded in the packaging system. The production and manipulation of lentiviral vectors should be carried out according to NIH guidelines for research involving recombinant DNA (<a href="http://oba.od.nih.gov/oba/rac/Guidelines/NIH_Guidelines.pdf" target="_blank">http://oba.od.nih.gov/oba/rac/Guidelines/NIH_Guidelines.pdf</a>). An approval from individual Institutional Biological and Chemical Safety Committee may be required before using lentiviral vectors. Lentiviral vectors are commonly produced by cotransfection of 293T cells with lentiviral transfer plasmid and the helper plasmids encoding the proteins required for vector packaging. Many lentiviral transfer plasmids and helper plasmids can be obtained from Addgene, a non-profit plasmid repository (<a href="http://www.addgene.org/" target="_blank">http://www.addgene.org/</a>). Some stable packaging cell lines have been developed, but these systems provide less flexibility and their packaging efficiency generally declines over time 14, 15. Commercially available transfection kits may support high efficiency of transfection 16, but they can be very expensive for large scale vector preparations. Calcium phosphate precipitation methods provide highly efficient transfection of 293T cells and thus provide a reliable and cost effective approach for lentiviral vector production.
In this protocol, we produce lentiviral vectors by cotransfection of 293T cells with four plasmids based on the calcium phosphate precipitation principle, followed by purification and concentration with ultracentrifugation through a 20% sucrose cushion. The vector titers are determined by fluorescence- activated cell sorting (FACS) analysis or by real time qPCR. The production and titration of lentiviral vectors in this protocol can be finished with 9 days. We provide an example of transducing these vectors into murine neocortical cultures containing both neurons and astrocytes. We demonstrate that lentiviral vectors support high efficiency of transduction and cell type-specific gene expression in primary cultured cells from CNS.

In this protocol we describe production, purification and titration of lentiviral vectors. We provide an example of lentiviral vector-mediated gene delivery in primary cultured neurons and astrocytes. Our methods may also apply to other cell types in vitro and in vivo.

Efficient gene delivery in the central nervous system (CNS) is important in studying gene functions, modeling neurological diseases and developing ...

Neuroscience

Transposon Mediated Integration of Plasmid DNA into the Subventricular Zone of Neonatal Mice to Generate Novel Models of Glioblastoma

An urgent need exists to test the contribution of new genes to the pathogenesis and progression of human glioblastomas (GBM), the most common primary brain tumor in adults with dismal prognosis. New potential therapies are rapidly emerging from the bench and require systematic testing in experimental models which closely reproduce the salient features of the human disease. Herein we describe in detail a method to induce new models of GBM with transposon-mediated integration of plasmid DNA into cells of the subventricular zone of neonatal mice. We present a simple way to clone new transposons amenable for genomic integration using the Sleeping Beauty transposon system and illustrate how to monitor plasmid uptake and disease progression using bioluminescence, histology and immuno-histochemistry. We also describe a method to create new primary GBM cell lines. Ideally, this report will allow further dissemination of the Sleeping Beauty transposon system among brain tumor researchers, leading to an in depth understanding of GBM pathogenesis and progression and to the timely design and testing of effective therapies for patients.

Here we describe an efficient and versatile protocol to induce, monitor and analyze novel glioblastomas (GBM) using transposon DNA injected into the ventricles of neonatal mice. Cells of the subventricular zone, which take up the plasmid, transform, proliferate and generate tumors with histo-pathological characteristics of human GBM.

An urgent need exists to test the contribution of new genes to the pathogenesis and progression of human glioblastomas (GBM), the most common primary ...

Medicine

Designing, Packaging, and Delivery of High Titer CRISPR Retro and Lentiviruses via Stereotaxic Injection

Replication defective lentiviruses or retroviruses are capable of stably integrating transgenes into the genome of an infected host cell. This technique has been widely used to encode fluorescent proteins, opto- or chemo-genetic controllers of cell activity, or heterologous expression of human genes in model organisms. These viruses have also successfully been used to deliver recombinases to relevant target sites in transgenic animals, or even deliver small hairpin or micro RNAs in order to manipulate gene expression. While these techniques have been fruitful, they rely on transgenic animals (recombinases) or frequently lack high efficacy and specificity (shRNA/miRNA). In contrast, the CRISPR/Cas system uses an exogenous Cas nuclease which targets specific sites in an organism's genome via an exogenous guide RNA in order to induce double stranded breaks in DNA. These breaks are then repaired by non-homologous end joining (NHEJ), producing insertion and deletion (indel) mutations that can result in deleterious missense or nonsense mutations. This manuscript provides detailed methods for the design, production, injection, and validation of single lenti/retro virus particles that can stably transduce neurons to express a fluorescent reporter, Cas9, and sgRNAs to knockout genes in a model organism.

The CRISPR/Cas9 system offers the potential to make targeted genome editing accessible and affordable to the scientific community. This protocol is intended to demonstrate how to create viruses that will knockout a gene of interest using the CRISPR/Cas9 system, and then inject them stereotaxically into the adult mouse brain.

Replication defective lentiviruses or retroviruses are capable of stably integrating transgenes into the genome of an infected host cell. This technique ...

Lentiviral Vector Preparation for In Vivo Gene Modulation 

Lentiviral Vector Preparation for Efficient Gene and MicroRNA Modulation of Peritoneal Cavity Tissue&#45;Resident Macrophages In Vivo in Mice

Peritoneal tissue-resident macrophages have broad functions in the maintenance of homeostasis and are involved in pathologies within local and neighboring tissues. Their functions are dictated by microenvironmental cues; thus, it is essential to investigate their behavior in an in vivo physiological niche. Currently, specific peritoneal macrophage-targeting methodologies employ whole-mouse transgenic models. Here, a protocol for effective in vivo modulation of mRNA and small RNA species (e.g., microRNA) expression in peritoneal macrophages using lentivirus particles is described. Lentivirus preparations were made in HEK293T cells and purified on a single sucrose layer. In vivo validation of lentivirus effectivity following intraperitoneal injection revealed predominant infection of macrophages restricted to local tissue. Targeting of peritoneal macrophages was successful during homeostasis and thioglycolate-induced peritonitis. The limitations of the protocol, including low-level inflammation induced by intraperitoneal delivery of lentivirus and time restrictions for potential experiments, are discussed. Overall, this study presents a quick and accessible protocol for the rapid assessment of gene function in peritoneal macrophages in vivo.

Author Spotlight: Exploring Macrophage Immunometabolism Through Lentiviral Vector&#45;Mediated Gene Manipulation

We demonstrate a step-by-step protocol for the investigation of gene function in peritoneal tissue-resident macrophages in vivo, using lentiviral vectors.

Peritoneal tissue-resident macrophages have broad functions in the maintenance of homeostasis and are involved in pathologies within local and neighboring ...

Laser-assisted Lentiviral Gene Delivery to Mouse Fertilized Eggs

Lentiviruses are efficient vectors for gene delivery to mammalian cells. Following transduction, the lentiviral genome is stably incorporated into the host chromosome and is passed on to progeny. Thus, they are ideal vectors for creation of stable cell lines, in vivo delivery of indicators, and transduction of single cell fertilized eggs to create transgenic animals. However, mouse fertilized eggs and early stage embryos are protected by the zona pellucida, a glycoprotein matrix that forms a barrier against lentiviral gene delivery. Lentiviruses are too large to penetrate the zona and are typically delivered by microinjection of viral particles into the perivitelline cavity, the space between the zona and the embryonic cells. The requirement for highly skilled technologists and specialized equipment has minimized the use of lentiviruses for gene delivery to mouse embryos. This article describes a protocol for permeabilizing the mouse fertilized eggs by perforating the zona with a laser. Laser-perforation does not result in any damage to embryos and allows lentiviruses to gain access to embryonic cells for gene delivery. Transduced embryos can develop into blastocyst in vitro, and if implanted in pseudopregnant mice, develop into transgenic pups. The laser used in this protocol is effective and easy to use. Genes delivered by lentiviruses stably incorporate into mouse embryonic cells and are germline transmittable. This is an alternative method for creation of transgenic mice that requires no micromanipulation and microinjection of fertilized eggs.

Mouse fertilized eggs and early stage embryos are protected by the zona pellucida, a glycoprotein matrix that forms a barrier against gene delivery. This article describes a protocol for perforating the zona with a laser to transduce embryonic cells with lentiviral vectors and to create transgenic mice.

Lentiviruses are efficient vectors for gene delivery to mammalian cells. Following transduction, the lentiviral genome is stably incorporated into the ...

Lentiviral Mediated Delivery of shRNAs to hESCs and NPCs Using Low-cost Cationic Polymer Polyethylenimine (PEI)

The current protocol describes the use of lentiviral particles for the delivery of short hairpin RNAs (shRNAs) to both human embryonic stem cells (hESCs) as well as neural progenitor cells (NPCs) derived from hESCs at high efficiency. Lentiviral particles were generated by co-transfecting HEK293T cells using entry vectors (carrying shRNAs) along with packaging plasmids (pAX and pMD2.G) using the low-cost cationic polymer polyethylenimine (PEI). Viral particles were concentrated using ultracentrifugation, which resulted in average titers above 5 x 107. Both hESCs and NPCs could be infected at high efficiencies using these lentiviral particles, as shown by puromycin selection and stable expression in hESCs, as well as transient GFP expression in NPCs. Furthermore, western blot analysis showed a significant reduction in the expression of genes targeted by shRNAs. In addition, the cells retained their pluripotency as well as differentiation potential, as evidenced by their subsequent differentiation into different lineages of CNS. The current protocol deals with the delivery of shRNAs; however, the same approach could be used for the ectopic expression of cDNAs for overexpression studies.

Lentiviral Mediated Delivery of shRNAs to hESCs and NPCs Using Low-cost Cationic Polymer Polyethylenimine PEI

Using the low-cost cationic polymer polyethylenimine (PEI), we produced lentiviral particles for stable expression of shRNAs in H9 human embryonic stem cells (hESCs) and transiently transduced H9-derived neural progenitor cells (NPCs) at high efficiency.

The current protocol describes the use of lentiviral particles for the delivery of short hairpin RNAs (shRNAs) to both human embryonic stem cells (hESCs) ...

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

Samenvatting

Meer video's verkennen