Name: production and purification of baculovirus for gene therapy application
Uploaded: 2018-04-09T13:30:00
Description: Watch this Scientific Journal Video about Production and Purification of Baculovirus for Gene Therapy Application at JoVE.com

This work is supported in part by the Start-Up funding from Cincinnati Children's Research Foundation (CCRF) to M.N. and Innovative Core Grant (ICG) supported by the National Center for Advancing Translational Sciences of the National Institutes of Health under Award Number UL1 TR001425. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

See Figure 1 for an illustration summarizing the protocol.
1. Purification of Baculovirus Plasmid DNA
<ol>
	<li>Grow bacterial culture containing baculoviral plasmid DNA (bacmid)14 in 200 mL of LB broth with antibiotics; kanamycin (50 &#181;g/mL), tetracycline (10 &#181;g/mL), and gentamycin (7 &#181;g/mL), for 16 h at 37 &#176;C on an orbital shaker setting at 300 rpm.</li>
	<li>Purify the bacmid DNA from the bacterial culture using stan...

<ol>
	<li>Ikonomou, L., Schneider, Y. J., Agathos, S. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insect+cell+culture+for+industrial+production+of+recombinant+proteins.">Insect cell culture for industrial production of recombinant proteins.</a> Appl Microbiol Biotechnol. 62 (1), 1-20 (2003).</li><li>Contreras-Gomez, A., Sanchez-Miron, A., Garcia-Camacho, F., Molina-Grima, E., Chisti, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+production+using+the+baculovirus-insect+cell+expression+system.">Protein production using the baculovirus-insect cell expression system.</a> Biotechnol Prog. 30 (1), 1-18 (2014).</li><li>Cox, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recombinant+protein+vaccines+produced+in+insect+cells.">Recombinant protein vaccines produced in insect cells.</a> Vaccine. 30 (10), 1759-1766 (2012).</li><li>Boyce, F. M., Bucher, N. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Baculovirus-mediated+gene+transfer+into+mammalian+cells.">Baculovirus-mediated gene transfer into mammalian cells.</a> Proc Natl Acad Sci U S A. 93 (6), 2348-2352 (1996).</li><li>Airenne, K. 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=Baculovirus:+an+insect-derived+vector+for+diverse+gene+transfer+applications.">Baculovirus: an insect-derived vector for diverse gene transfer applications.</a> Mol Ther. 21 (4), 739-749 (2013).</li><li>Sung, L. Y., 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=Efficient+gene+delivery+into+cell+lines+and+stem+cells+using+baculovirus.">Efficient gene delivery into cell lines and stem cells using baculovirus.</a> Nat Protoc. 9 (8), 1882-1899 (2014).</li><li>Zeng, J., Du, J., Lin, J., Bak, X. Y., Wu, C., Wang, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-efficiency+transient+transduction+of+human+embryonic+stem+cell-derived+neurons+with+baculoviral+vectors.">High-efficiency transient transduction of human embryonic stem cell-derived neurons with baculoviral vectors.</a> Mol Ther. 17 (9), 1585-1593 (2009).</li><li>Zeng, J., Du, J., Zhao, Y., Palanisamy, N., Wang, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Baculoviral+vector-mediated+transient+and+stable+transgene+expression+in+human+embryonic+stem+cells.">Baculoviral vector-mediated transient and stable transgene expression in human embryonic stem cells.</a> Stem Cells. 25 (4), 1055-1061 (2007).</li><li>Wu, C., Soh, K. Y., Wang, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ion-exchange+membrane+chromatography+method+for+rapid+and+efficient+purification+of+recombinant+baculovirus+and+baculovirus+gp64+protein.">Ion-exchange membrane chromatography method for rapid and efficient purification of recombinant baculovirus and baculovirus gp64 protein.</a> Hum Gene Ther. 18 (7), 665-672 (2007).</li><li>Grein, T. A., Michalsky, R., Vega Lopez, M., Czermak, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+a+recombinant+baculovirus+of+Autographa+californica+M+nucleopolyhedrovirus+by+ion+exchange+membrane+chromatography.">Purification of a recombinant baculovirus of Autographa californica M nucleopolyhedrovirus by ion exchange membrane chromatography.</a> J Virol Methods. 183 (2), 117-124 (2012).</li><li>Nasimuzzaman, M., Lynn, D., van der Loo, J. C., Malik, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+baculovirus+vectors+using+heparin+affinity+chromatography.">Purification of baculovirus vectors using heparin affinity chromatography.</a> Mol Ther Methods Clin Dev. 3, 16071 (2016).</li><li>Makkonen, K. E., Turkki, P., Laakkonen, J. P., Yla-Herttuala, S., Marjomaki, V., Airenne, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=6-o-+and+N-sulfated+syndecan-1+promotes+baculovirus+binding+and+entry+into+Mammalian+cells.">6-o- and N-sulfated syndecan-1 promotes baculovirus binding and entry into Mammalian cells.</a> J Virol. 87 (20), 11148-11159 (2013).</li><li>Nasimuzzaman, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heparan+sulfate+in+baculovirus+binding+and+entry+of+mammalian+cells.">Heparan sulfate in baculovirus binding and entry of mammalian cells.</a> J Virol. 88 (8), 4607-4608 (2014).</li><li>Urabe, 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=Scalable+generation+of+high-titer+recombinant+adeno-associated+virus+type+5+in+insect+cells.">Scalable generation of high-titer recombinant adeno-associated virus type 5 in insect cells.</a> J Virol. 80 (4), 1874-1885 (2006).</li><li>Feliciello, I., Chinali, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+modified+alkaline+lysis+method+for+the+preparation+of+highly+purified+plasmid+DNA+from+Escherichia+coli.">A modified alkaline lysis method for the preparation of highly purified plasmid DNA from Escherichia coli.</a> Anal Biochem. 212 (2), 394-401 (1993).</li><li>Nasimuzzaman, M., Persons, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+Membrane-associated+heparan+sulfate+is+a+receptor+for+prototype+foamy+virus+in+human,+monkey,+and+rodent+cells.">Cell Membrane-associated heparan sulfate is a receptor for prototype foamy virus in human, monkey, and rodent cells.</a> Mol Ther. 20 (6), 1158-1166 (2012).</li><li>Earl, P. L., Americo, J. L., Moss, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+use+of+a+vaccinia+virus+neutralization+assay+based+on+flow+cytometric+detection+of+green+fluorescent+protein.">Development and use of a vaccinia virus neutralization assay based on flow cytometric detection of green fluorescent protein.</a> J Virol. 77 (19), 10684-10688 (2003).</li><li>Kim, Y. K., 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=Suppression+of+tumor+growth+in+xenograft+model+mice+by+programmed+cell+death+4+gene+delivery+using+folate-PEG-baculovirus.">Suppression of tumor growth in xenograft model mice by programmed cell death 4 gene delivery using folate-PEG-baculovirus.</a> Cancer Gene Ther. 17 (11), 751-760 (2010).</li><li>Stanbridge, L. J., Dussupt, V., Maitland, N. 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The protocol presented here describes the production of baculovirus in Sf9 cells in suspension culture and purification of baculovirus using a heparin affinity chromatography. The parameters used in this protocol maximize the yield and minimize the inactivation of infectious baculovirus. The protocol provided here shows a significantly improved recovery of baculovirus particles as compared to recoveries achieved by others9.
Due to broad host range and tissue tropism, se...

Baculovirus is primarily used for the production of recombinant proteins and vaccines in lepidopteran Spodoptera fugiperda (Sf)9 insect cells by using recombinant Autographa californica multicapsid nuclear polyhedrosis virus (AcMNPV)1,2,3,4. More recently, it is emerging as a promising vector for gene therapy application5. It is known to have a broad host and tissue tropism, infects both quiescent and proliferating cells, is non-pathogenic, and does not integrate into the host chromosome4,5,6. Moreover, baculovirus can be produced in serum-free suspension culture which is scalable and allows for closed system processing for future clinical production1.
The purity of baculovirus particles is important for achieving effective transduction while minimizing cytotoxicity7,8,9. Baculovirus can be concentrated by ultracentrifugation or tangential flow filtration (TFF) with limited impact on its infectivity. However, these procedures not only concentrate virus particles but also cellular debris and proteins from Sf9 culture, which can be toxic in vitro (personal observation) and may induce inflammation or an immune response when used in vivo. To avoid this, especially when using highly concentrated virus stocks, infectious baculovirus needs to be purified and separated from contaminating particles.
Several methods have been reported for the purification and concentration of baculovirus vectors10,11,12. Of the available approaches, heparin affinity chromatography allows for a single-step high level of purification with the low concentration of contaminating proteins12. The method is based on the identification of heparan sulfate as the receptor for baculovirus13,14. After loading Sf9 cell supernatant onto the column and binding of baculovirus, the column can be washed with physiologic (isotonic) buffer to remove unbound or loosely bound contaminating particles. Since the binding to heparin is reversible, baculovirus particles can be eluted with a high salt buffer, which is diluted immediately to physiologic (isotonic) salt concentration to prevent inactivation by osmotic shock12. Moreover, the production of baculovirus, as well as capture on and elution from the chromatography column, can be performed using a closed-system process which is compatible with current good manufacturing practices (cGMP).
Here, we provide a detailed protocol for the manufacture, purification, and concentration of infectious baculovirus using affinity chromatography and centrifugation. Briefly, we produce baculovirus by transfection of Sf9 cells with a baculovirus plasmid DNA in adherent culture and further expand the infectious baculovirus in serum-free suspension culture. We purify baculovirus using heparin affinity chromatography and use ultracentrifugation as the final step to highly concentrate the vector for gene therapy application.

<table border="0" cellpadding="0" cellspacing="0" style="width:784px;" width="784">
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	</colgroup>
	<tbody>
		<tr height="16">
			<td height="16" style="height:16px;width:257px;">Akta Avant 150</td>
			<td style="width:204px;">GE Healthcare</td>
			<td style="width:156px;">28976337</td>
			<td style="width:167px;">Chromatography system</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:257px;">POROS Heparin 50 &#181;m Column</td>
			<td>ThermoFisher Scientific</td>
			<td>4333414</td>
			<td>Heparin column</td>
		</tr>
		<tr height="18">
			<td height="18" style="height:18px;">Ultracentrifuge</td>
			<td>Beckman-Coulter</td>
			<td>Non-catalog item</td>
			<td>Concentrates virus at high-speed</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">Polyallomer Ultracentrifuge tube</td>
			<td>Beckman-Coulter</td>
			<td>326823</td>
			<td>Concentrates virus at high-speed</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">MaxQ 8000 orbital shaker incubator&#160;</td>
			<td>ThermoFisher Scientific</td>
			<td>Non-catalog item</td>
			<td>Shaker for suspension culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">250 mL Erlenmeyer flasks</td>
			<td>ThermoFisher Scientific</td>
			<td>238071</td>
			<td>Flask for suspension culture</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1 L Erlenmeyer flasks</td>
			<td>ThermoFisher Scientific</td>
			<td>238072</td>
			<td>Flask for suspension culture</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Microscope (Olympus CKX41)</td>
			<td>Olympus</td>
			<td>Non-catalog item</td>
			<td>Cell monitoring and counting&#160;</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Table top centrifuge</td>
			<td>ThermoFisher Scientific</td>
			<td>75253839/433607</td>
			<td>For clarification of Baculovirus supernatant</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">50 ml Conical tube</td>
			<td>ThermoFisher Scientific</td>
			<td>14-959-49A</td>
			<td>For collection of Baculovirus supernatant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">6-well plate</td>
			<td>ThermoFisher Scientific</td>
			<td>07-200-80</td>
			<td>Tissue culture treated plate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10-cm plate</td>
			<td>ThermoFisher Scientific</td>
			<td>08-772E</td>
			<td>Tissue culture treated plate</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:257px;">Stericup-HV, 0.45&#160;&#181;m, PVDF</td>
			<td>EMD-Millipore</td>
			<td>SCHVU05RE</td>
			<td>Filtration unit</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Kanamycin</td>
			<td>ThermoFisher Scientific</td>
			<td>15160-054</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tetracycline</td>
			<td>Sigma-Aldrich</td>
			<td>T7660</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Gentamycin</td>
			<td>ThermoFisher Scientific</td>
			<td>15750-060</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bac-to-Bac Vector Kit</td>
			<td>ThermoFisher Scientific</td>
			<td>10360-014</td>
			<td>Baculovirus expression system</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DH10B-T1R&#160; Competent cell</td>
			<td>ThermoFisher Scientific</td>
			<td>12331-013</td>
			<td>Competent cell for bacmid</td>
		</tr>
		<tr height="16">
			<td height="16" style="height:16px;">TE buffer</td>
			<td>In-house</td>
			<td>Non-catalog item</td>
			<td>10 mM Tris-HCl, 1 mM EDTA, pH 8.0.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Plasmid Maxiprep kit</td>
			<td>ThermoFisher Scientific</td>
			<td>K2100-06</td>
			<td>For bacmid purification</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Sf9 Cells</td>
			<td>ThermoFisher Scientific</td>
			<td>11496-015</td>
			<td>Insect cells</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">Grace&#8217;s Insect Cell Culture Medium</td>
			<td>ThermoFisher Scientific</td>
			<td>11605-094</td>
			<td>Transfection medium</td>
		</tr>
		<tr height="16">
			<td height="16" style="height:16px;">PBS</td>
			<td>ThermoFisher Scientific</td>
			<td>20012227</td>
			<td>Washing cells, diluting samples</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:257px;">HyClone SFX-Insect cell media</td>
			<td>GE Healthcare</td>
			<td>SH30278.02</td>
			<td>Serum-free insect cell growth medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">Benzonase Nuclease</td>
			<td>Sigma-Aldrich</td>
			<td>E1014</td>
			<td>Enzyme to degrade DNA and RNA</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Baculovirus plasmid (bacmid) DNA</td>
			<td>In-house</td>
			<td>Non-catalog item</td>
			<td>Bacmid for Baculovirus Production</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cellfectin II&#160;</td>
			<td>ThermoFisher Scientific</td>
			<td>10362</td>
			<td>Transfection reagent for insect cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bovine serum albumin (BSA)</td>
			<td>Sigma-Aldrich</td>
			<td>A4737</td>
			<td>Stabilizes Baculovirus</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cryovial</td>
			<td>Thomas Scientific</td>
			<td>1222C24</td>
			<td>For storage of Baculovirus</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HT1080 cell line</td>
			<td>ATCC</td>
			<td style="width:156px;">CCL-121</td>
			<td>Fibroblast cell line</td>
		</tr>
		<tr height="18">
			<td height="18" style="height:18px;">DMEM</td>
			<td>Sigma-Aldrich</td>
			<td>D6429</td>
			<td>Growth media for cell lines</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Wash buffer</td>
			<td>In-house</td>
			<td>Non-catalog item</td>
			<td>20 mmol/l phosphate buffer containing 150 mmol/l sodium chloride</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Elution buffer</td>
			<td>In-house</td>
			<td>Non-catalog item</td>
			<td>20 mmol/l phosphate buffer containing 1.5 mol/l sodium chloride</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Column cleaning buffer</td>
			<td>In-house</td>
			<td>Non-catalog item</td>
			<td>20 mmol/l phosphate buffer containing 2.0 mol/l sodium chloride</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sterile water</td>
			<td>In-house</td>
			<td>Non-catalog item</td>
			<td>For Akta Avant cleaning</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Sodium hydroxide</td>
			<td>Sigma-Aldrich</td>
			<td>1.09137</td>
			<td>For Akta Avant cleaning</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ethanol</td>
			<td>Sigma-Aldrich</td>
			<td>E7073</td>
			<td>For Akta Avant cleaning</td>
		</tr>
	</tbody>
</table>

production and purification of baculovirus for gene therapy application

Baculovirus has traditionally been used for the production of recombinant protein and vaccine. However, more recently, baculovirus is emerging as a promising vector for gene therapy application. Here, baculovirus is produced by transient transfection of the baculovirus plasmid DNA (bacmid) in an adherent culture of Sf9 cells. Baculovirus is subsequently expanded in Sf9 cells in a serum-free suspension culture until the desired volume is obtained. It is then purified from the culture supernatant using heparin affinity chromatography. Virus supernatant is loaded onto the heparin column which binds baculovirus particles in the supernatant due to the affinity of heparin for baculovirus envelop glycoprotein. The column is washed with a buffer to remove contaminants and baculovirus is eluted from the column with a high-salt buffer. The eluate is diluted to an isotonic salt concentration and baculovirus particles are further concentrated using ultracentrifugation. Using this method, baculovirus can be concentrated up to 500-fold with a 25% recovery of infectious particles. Although the protocol described here demonstrates the production and purification of the baculovirus from cultures up to 1 L, the method can be scaled-up in a closed-system suspension culture to produce a clinical-grade vector for gene therapy application.

The protocol presented is in a flow diagram (Figure 1). Steps include the transient transfection of Sf9 cells with bacmid DNA to produce baculovirus in adherent culture in a plate, the subsequent amplification in serum-free suspension culture, nuclease treatment and clarification by centrifugation and filtration, and the purification using the heparin affinity chromatography followed by concentration with ultracentrifugation.
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Watch this Scientific Journal Video about Production and Purification of Baculovirus for Gene Therapy Application at JoVE.com

Production and Purification of Baculovirus for Gene Therapy Application

In this protocol, baculovirus is produced by transient transfection of baculovirus plasmid into Sf9 cells and amplified in a serum-free suspension culture. The supernatant is purified by heparin affinity chromatography and further concentrated by ultracentrifugation. This protocol is useful for the production and purification of baculovirus for gene therapy application.

Baculovirus has traditionally been used for the production of recombinant protein and vaccine. However, more recently, baculovirus is emerging as a ...

The overall goal of this protocol is to produce baculovirus in insect cell culture, purify with heparin affinity chromatography and to concentrate with ultracentrifugation. The final product can be used for in vivo gene transfer or as a vaccine. This method can help researchers who want to use purified and concentrated baculovirus for gene therapy application.Baculovirus particles may contain cellular debris and proteins from Sf9 culture which may induce inflammation or an immune response when used in people. To avoid toxicity, baculovirus is purified and separated from contaminating particles. Some parts of the procedure will be demonstrated by Marilou Narciso, a research assistant from my laboratory.To set up the suspension culture, seed 50 milliliters of Sf9 cells in insect culture medium into a 250 milliliters polycarbonate Erlenmeyer flask. And place the flask in an orbital shaker incubator. Add two milliliters of P2 baculovirus stock to the cells, and shake at 135rpm while maintaining a constant temperature of 28 degrees Celsius.Four days after infection, the entire Sf9 cell population will show cytopathic effect, which is an indication of high titer baculovirus production. Sequentially amplify the baculovirus until the desired volume is obtained, by increasing the volume of cell culture in each subsequent infection 10 to 50-fold. After centrifuging the final baculovirus supernatant for 30 minutes at 1000g and four degrees Celsius to separate the course cellular debris, transfer the supernatant to a clean vessel and treat with nuclease at 50 units per milliliter for two hours at 37 degrees Celsius to digest the genomic DNA and RNA.Finally, filter the viral supernatant through a 0.45 micron filtration unit. Set up the chromatography system by placing the wash buffer line from inlet port A1 into a bottle containing at least 500 milliliters of wash buffer. Use the A2 buffer inlet port for loading the elution buffer.Insert several 50 milliliters conical tubes into the fraction collector to collect the column pass-through baculovirus supernatant, the wash buffer, and the eluded baculovirus. Equilibrate the heparin column with five 7.9 milliliters column volumes of wash buffer at a linear flow rate of seven milliliters per minute. Load 250 milliliters of baculovirus supernatant, onto the heparin column using the sample pump of the inlet sample port at a linear flow rate of two milliliters per minute.Then run 10 column volumes of wash buffer through the heparin column at a linear flow rate of two milliliters per minute until the ultraviolet absorbance curve has returned to baseline and become stable. Elude the baculovirus particles from the heparin column with five column volumes of elution buffer at a linear flow rate of four milliliters per minute. Watch for a sharp elution peak of protein on the chromatogram when the baculovirus particles dissociate from the heparin column.Post elution, immediately dilute the eluded baculovirus supernatant 10-fold using 20 millimeter sodium phosphate buffer to prevent an activation of baculovirus particles from osmotic shock during subsequent centrifugation. Begin the concentration procedure by adding an equal volume of the baculovirus supernatant to autoclave to ultracentrifuge tubes. Measure the tube weight by a balance and adjust the weight of the contents of the ultracentrifuge tubes with sterile PBS.Place each of the ultracentrifuge tubes in an opposing bucket of the SW 32 Ti rotor and run the ultracentrifuge at 80, 000g for 90 minutes at four degrees Celsius. Following centrifugation, open the ultracentrifuge tubes in a tissue culture hood and aseptically aspirate the liquid without removing the baculovirus pellet. Add 200 microliters of PBS containing 0.5%BSA and resuspend each pellet by vigorously pipetting up and down.Finally, transfer 50 to 100 microliter aliquots of the concentrated baculovirus to cryovials and store at negative 80 degrees Celsius. Baculovirus titers were estimated by infection of HT1080 cells. The line connected by diamonds shows the total infectious units of baculovirus in each fraction.Baculovirus particles strongly bind to the heparin column, therefore no significant amount of virus is detected in the column wash buffer. A sharp peak of protein is observed during elution, which corresponds to the baculovirus fraction. The parameters used in this protocol maximize the yield and minimize the inactivation of infectious baculovirus particles.This protocol can be used for purification of other viruses and proteins that have an affinity for the heparin.

production-purification-baculovirus-for-gene-therapy

Research

JoVE Journal

Genetics

Genetic Engineering of an Unconventional Yeast for Renewable Biofuel and Biochemical Production

Yarrowia lipolytica is a non-pathogenic, dimorphic and strictly aerobic yeast species. Owing to its distinctive physiological features and metabolic characteristics, this unconventional yeast is not only a good model for the study of the fundamental nature of fungal differentiation but is also a promising microbial platform for biochemical production and various biotechnological applications, which require extensive genetic manipulations. However, genetic manipulations of Y. lipolytica have been limited due to the lack of an efficient and stable genetic transformation system as well as very high rates of non-homologous recombination that can be mainly attributed to the KU70 gene. Here, we report an easy and rapid protocol for the efficient genetic transformation and for gene deletion in Y. lipolytica Po1g. First, a protocol for the efficient transformation of exogenous DNA into Y. lipolytica Po1g was established. Second, to achieve the enhanced double-crossover homologous recombination rate for further deletion of target genes, the KU70 gene was deleted by transforming a disruption cassette carrying 1 kb homology arms. Third, to demonstrate the enhanced gene deletion efficiency after deletion of the KU70 gene, we individually deleted 11 target genes encoding alcohol dehydrogenase and alcohol oxidase using the same procedures on the KU70 knockout platform strain. It was observed that the rate of precise homologous recombination increased substantially from less than 0.5% for deletion of the KU70 gene in Po1g to 33%-71% for the single gene deletion of the 11 target genes in Po1g KU70&#916;. A replicative plasmid carrying the hygromycin B resistance marker and the Cre/LoxP system was constructed, and the selection marker gene in the yeast knockout strains was eventually removed by expression of Cre recombinase to facilitate multiple rounds of targeted genetic manipulations. The resulting single-gene deletion mutants have potential applications in biofuel and biochemical production.

We herein report methods on the molecular genetic manipulation of the Yarrowia lipolytica Po1g strain for improved gene deletion efficiency. The resulting engineered Y. lipolytica strains have potential applications in biofuel and biochemical production.

Yarrowia lipolytica is a non-pathogenic, dimorphic and strictly aerobic yeast species. Owing to its distinctive physiological features and metabolic ...

Single-cell Gene Expression Profiling Using FACS and qPCR with Internal Standards

Gene expression measurements from bulk populations of cells can obscure the considerable transcriptomic variation of individual cells within those populations. Single-cell gene expression measurements can help assess the role of noise in gene expression, identify correlations in the expression of pairs of genes, and reveal subpopulations of cells that respond differently to a stimulus. Here, we describe a procedure to measure the expression of up to 96 genes in single mammalian cells isolated from a population growing in tissue culture. Cells are sorted into lysis buffer by fluorescence-activated cell sorting (FACS), and the mRNA species of interest are reverse-transcribed and amplified. Gene expression is then measured using a microfluidic real-time PCR machine, which performs up to 96 qPCR assays on up to 96 samples at a time. We also describe the generation and use of PCR amplicon standards to enable the estimation of the absolute number of each transcript. Compared with other methods of measuring gene expression in single cells, this approach allows for the quantification of more distinct transcripts than RNA FISH at a lower cost than RNA-Seq.

We describe a method to sort single mammalian cells and to quantify the expression of up to 96 target genes of interest in each cell. This method includes the use of internal qPCR standards to enable the estimation of absolute transcript counts.

Gene expression measurements from bulk populations of cells can obscure the considerable transcriptomic variation of individual cells within those ...

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 ...

Efficient Production and Identification of CRISPR/Cas9-generated Gene Knockouts in the Model System Danio rerio

Characterization of the clustered, regularly interspaced, short, palindromic repeat (CRISPR) system of Streptococcus pyogenes has enabled the development of a customizable platform to rapidly generate gene modifications in a wide variety of organisms, including zebrafish. CRISPR-based genome editing uses a single guide RNA (sgRNA) to target a CRISPR-associated (Cas) endonuclease to a genomic DNA (gDNA) target of interest, where the Cas endonuclease generates a double-strand break (DSB). Repair of DSBs by error-prone mechanisms lead to insertions and/or deletions (indels). This can cause frameshift mutations that often introduce a premature stop codon within the coding sequence, thus creating a protein-null allele. CRISPR-based genome engineering requires only a few molecular components and is easily introduced into zebrafish embryos by microinjection. This protocol describes the methods used to generate CRISPR reagents for zebrafish microinjection and to identify fish exhibiting germline transmission of CRISPR-modified genes. These methods include in vitro transcription of sgRNAs, microinjection of CRISPR reagents, identification of indels induced at the target site using a PCR-based method called a heteroduplex mobility assay (HMA), and characterization of the indels using both a low throughput and a powerful next-generation sequencing (NGS)-based approach that can analyze multiple PCR products collected from heterozygous fish. This protocol is streamlined to minimize both the number of fish required and the types of equipment needed to perform the analyses. Furthermore, this protocol is designed to be amenable for use by laboratory personal of all levels of experience including undergraduates, enabling this powerful tool to be economically employed by any research group interested in performing CRISPR-based genomic modification in zebrafish.

Efficient Production and Identification of CRISPR/Cas9-generated Gene Knockouts in the Model System Danio rerio

Targeted genome editing in the model system Danio rerio (zebrafish) has been greatly facilitated by the emergence of CRISPR-based approaches. Herein, we describe a streamlined, robust protocol for generation and identification of CRISPR-derived nonsense alleles that incorporates the heteroduplex mobility assay and identification of mutations using next-generation sequencing.

Characterization of the clustered, regularly interspaced, short, palindromic repeat (CRISPR) system of Streptococcus pyogenes has enabled the development ...

Highly Efficient Gene Disruption of Murine and Human Hematopoietic Progenitor Cells by CRISPR/Cas9

Advances in the hematopoietic stem cell (HSCs) field have been aided by methods to genetically engineer primary progenitor cells as well as animal models. Complete gene ablation in HSCs required the generation of knockout mice from which HSCs could be isolated, and gene ablation in primary human HSCs was not possible. Viral transduction could be used for knock-down approaches, but these suffered from variable efficacy. In general, genetic manipulation of human and mouse hematopoietic cells was hampered by low efficiencies and extensive time and cost commitments. Recently, CRISPR/Cas9 has dramatically expanded the ability to engineer the DNA of mammalian cells. However, the application of CRISPR/Cas9 to hematopoietic cells has been challenging, mainly due to their low transfection efficiencies, the toxicity of plasmid-based approaches and the slow turnaround time of virus-based protocols.
A rapid method to perform CRISPR/Cas9-mediated gene editing in murine and human hematopoietic stem and progenitor cells with knockout efficiencies of up to 90% is provided in this article. This approach utilizes a ribonucleoprotein (RNP) delivery strategy with a streamlined three-day workflow. The use of Cas9-sgRNA RNP allows for a hit-and-run approach, introducing no exogenous DNA sequences in the genome of edited cells and reducing off-target effects. The RNP-based method is fast and straightforward: it does not require cloning of sgRNAs, virus preparation or specific sgRNA chemical modification. With this protocol, scientists should be able to successfully generate knockouts of a gene of interest in primary hematopoietic cells within a week, including downtimes for oligonucleotide synthesis. This approach will allow a much broader group of users to adapt this protocol for their needs.

A protocol for fast CRISPR/Cas9-mediated gene disruption in mouse and human primary hematopoietic cells is described in this article. Cas9-sgRNA ribonucleoproteins are introduced via electroporation with sgRNAs generated through in vitro transcription and commercial Cas9. High editing efficiencies are achieved with limited time and financial cost.

Advances in the hematopoietic stem cell (HSCs) field have been aided by methods to genetically engineer primary progenitor cells as well as animal models. ...

Preparation and Gene Modification of Nonhuman Primate Hematopoietic Stem and Progenitor Cells

Hematopoietic stem and progenitor cell (HSPC) transplantation has been a cornerstone therapy for leukemia and other cancers for nearly half a century, underlies the only known cure of human immunodeficiency virus (HIV-1) infection, and shows immense promise in the treatment of genetic diseases such as beta thalassemia. Our group has developed a protocol to model HSPC gene therapy in nonhuman primates (NHPs), allowing scientists to optimize many of the same reagents and techniques that are applied in the clinic. Here, we describe methods for purifying CD34+ HSPCs and long-term persisting hematopoietic stem cell (HSC) subsets from primed bone marrow (BM). Identical techniques can be employed for the purification of other HSPC sources (e.g., mobilized peripheral blood stem cells [PBSCs]). Outlined is a 2 day protocol in which cells are purified, cultured, modified with lentivirus (LV), and prepared for infusion back into the autologous host. Key readouts of success include the purity of the CD34+ HSPC population, the ability of purified HSPCs to form morphologically distinct colonies in semisolid media, and, most importantly, gene modification efficiency. The key advantage to HSPC gene therapy is the ability to provide a source of long-lived cells that give rise to all hematopoietic cell types. As such, these methods have been used to model therapies for cancer, genetic diseases, and infectious diseases. In each case, therapeutic efficacy is established by enhancing the function of distinct HSPC progeny, including red blood cells, T cells, B cells, and/or myeloid subsets. The methods to isolate, modify, and prepare HSPC products are directly applicable and translatable to multiple diseases in human patients.

The goal of this protocol is to isolate nonhuman primate CD34+ cells from primed bone marrow, to gene-modify these cells with lentiviral vectors, and to prepare a product for infusion into the autologous host. The total protocol length is approximately 48 h.

Hematopoietic stem and progenitor cell (HSPC) transplantation has been a cornerstone therapy for leukemia and other cancers for nearly half a century, ...

In vivo Application of the REMOTE-control System for the Manipulation of Endogenous Gene Expression

Here we describe a protocol for implementing the REMOTE-control system (Reversible Manipulation of Transcription at Endogenous loci), which allows for reversible and tunable expression control of an endogenous gene of interest in living model systems. The REMOTE-control system employs enhanced lac repression and tet activation systems to achieve down- or upregulation of a target gene within a single biological system. Tight repression can be achieved from repressor binding sites flexibly located far downstream of a transcription start site by inhibiting transcription elongation. Robust upregulation can be attained by enhancing the transcription of an endogenous gene by targeting tet transcriptional activators to the cognate promoter. This reversible and tunable expression control can be applied and withdrawn repeatedly in organisms. The potency and versatility of the system, as demonstrated for endogenous Dnmt1 here, will allow more precise in vivo functional analyses by enabling investigation of gene function at various expression levels and by testing the reversibility of a phenotype.

This protocol outlines the steps needed to generate a model system in which the transcription of an endogenous gene of interest can be conditionally controlled in live animals or cells using enhanced lac repressor and/or tet activator systems.

Here we describe a protocol for implementing the REMOTE-control system (Reversible Manipulation of Transcription at Endogenous loci), which allows for ...

Transforming, Genome Editing and Phenotyping the Nitrogen-fixing Tropical Cannabaceae Tree Parasponia andersonii

Parasponia andersonii is a fast-growing tropical tree that belongs to the Cannabis family (Cannabaceae). Together with 4 additional species, it forms the only known non-legume lineage able to establish a nitrogen-fixing nodule symbiosis with rhizobium. Comparative studies between legumes and P. andersonii could provide valuable insight into the genetic networks underlying root nodule formation. To facilitate comparative studies, we recently sequenced the P. andersonii genome and established Agrobacterium tumefaciens-mediated stable transformation and CRISPR/Cas9-based genome editing. Here, we provide a detailed description of the transformation and genome editing procedures developed for P. andersonii. In addition, we describe procedures for the seed germination and characterization of symbiotic phenotypes. Using this protocol, stable transgenic mutant lines can be generated in a period of 2-3 months. Vegetative in vitro propagation of T0 transgenic lines allows phenotyping experiments to be initiated at 4 months after A. tumefaciens co-cultivation. Therefore, this protocol takes only marginally longer than the transient Agrobacterium rhizogenes-based root transformation method available for P. andersonii, though offers several clear advantages. Together, the procedures described here permit P. andersonii to be used as a research model for studies aimed at understanding symbiotic associations as well as potentially other aspects of the biology of this tropical tree.

Transforming, Genome Editing and Phenotyping the Nitrogen-fixing Tropical Cannabaceae Tree Parasponia andersonii

Parasponia andersonii is a fast-growing tropical tree that belongs to the Cannabis family (Cannabaceae) and can form nitrogen-fixing root nodules in association with the rhizobium. Here, we describe a detailed protocol for reverse genetic analyses in P. andersonii based on Agrobacterium tumefaciens-mediated stable transformation and CRISPR/Cas9-based genome editing.

Parasponia andersonii is a fast-growing tropical tree that belongs to the Cannabis family (Cannabaceae). Together with 4 additional species, it forms the ...

Characterization of Functionally Associated miRNAs in Glioblastoma and their Engineering into Artificial Clusters for Gene Therapy

The biological relevance of microRNAs (miRNAs) in health and disease significantly relies on specific combinations of many simultaneously deregulated miRNAs rather than the action of a single miRNA. The characterization of these specific miRNAs modules is a fundamental step in maximizing their use in therapy. This is extremely relevant because their combinatorial attributes can be practically exploited. Described here is a method to define a specific miRNA signature relevant to the control of oncogenic chromatin repressors in glioblastoma. The approach first defines a general group of miRNAs that are deregulated in tumors in comparison to normal tissue. The analysis is further refined by differential culture conditions, underscoring a subgroup of miRNAs that are co-expressed simultaneously during specific cellular states. Finally, the miRNAs that satisfy these filters are combined into an artificial polycistronic transgenes, which is based on a scaffold of naturally existing miRNA clusters genes, then used for overexpression of these miRNA modules into receiving cells.

Described here is a protocol for characterizing modules of biologically synergistic miRNAs and their assembly into short transgenes, which allows simultaneous overexpression for gene therapy applications.

The biological relevance of microRNAs (miRNAs) in health and disease significantly relies on specific combinations of many simultaneously deregulated ...

Use of Freeze-thawed Embryos for High-efficiency Production of Genetically Modified Mice

The use of genetically modified (GM) mice has become crucial for understanding gene function and deciphering the underlying mechanisms of human diseases. The CRISPR/Cas9 system allows researchers to modify the genome with unprecedented efficiency, fidelity, and simplicity. Harnessing this technology, researchers are seeking a rapid, efficient, and easy protocol for generating GM mice. Here we introduce an improved method for cryopreservation of one-cell embryos that leads to a higher developmental rate of the freeze-thawed embryos. By combining it with optimized electroporation conditions, this protocol allows for the generation of knockout and knock-in mice with high efficiency and low mosaic rates within a short time. Furthermore, we show a step-by-step explanation of our optimized protocol, covering CRISPR reagent preparation, in vitro fertilization, cryopreservation and thawing of one-cell embryos, electroporation of CRISPR reagents, mouse generation, and genotyping of the founders. Using this protocol, researchers should be able to prepare GM mice with unparalleled ease, speed, and efficiency.

Here, we present a modified method for cryopreservation of one-cell embryos as well as a protocol that couples the use of freeze-thawed embryos and electroporation for the efficient generation of genetically modified mice.

The use of genetically modified (GM) mice has become crucial for understanding gene function and deciphering the underlying mechanisms of human diseases. ...