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 standard alkaline lysis protocol15, and dissolve the bacmid in TE buffer.</li>
</ol>
2. Production of Baculovirus
<ol>
	<li>Culture Sf9 cells in an orbital shaker incubator at 135 rpm and 28 &#176;C in a polycarbonate Erlenmeyer flask containing serum-free insect culture medium1,2,3. 
	Note: If Sf9 cells are thawed from frozen stock, allow at least two weeks for the cells to recover and enter the exponential phase of growth.</li>
	<li>Count the Sf9 cells harvested at exponential phase of growth with a hemocytometer after staining with Trypan blue. Seed 1 x 106 viable Sf9 cells per well in a 6-well tissue culture-treated plate in 2 mL of medium. Allow the cell to attach for 1 h at 28 &#176;C in an incubator.</li>
	<li>Replace the growth medium with 1 mL of serum-free unsupplemented Grace&#39;s insect culture medium.</li>
	<li>Transfect the bacmid DNA into the Sf9 cells using insect cell transfection reagent.
	<ol>
		<li>Mix 8 &#181;L insect cell transfection reagent with 100 &#181;L of Grace&#39;s Insect medium.</li>
		<li>Dilute 2 &#181;g of bacmid DNA into 100 &#181;L of unsupplemented insect medium, and mix gently by vortexing.</li>
		<li>Combine the diluted DNA with the diluted insect cell transfection reagent, and mix gently. Incubate for 15 to 30 min at room temperature.</li>
		<li>Add the DNA-lipid mixture dropwise onto the cells. Incubate at 28 &#176;C in an incubator for approximately 5 h.</li>
	</ol>
	</li>
	<li>Remove the transfection mixture from the cells and wash the cells with 2 mL of PBS.</li>
	<li>Add 2 mL of insect culture medium and continue to culture at 28 &#176;C in an incubator without changing the media. 
	Note: If the bacmid contains a fluorescent protein, its expression can be detected in cells 48 h post-transfection. Baculovirus is produced by transfected Sf9 cells and secreted into the culture medium which subsequently infects the untransfected cells. The entire cell population becomes infected with baculovirus in 5 days and shows signs of viral infection, also called cytopathic effect. These include increased cell diameter, vacuoles/granules in the cytoplasm, cessation of cell growth and dead or lysed cells are present in cell culture. However, if the transfection or infection efficiency is not optimal, the culture may not show an obvious cytopathic effect. The cytopathic effect can be visualized with an inverted phase microscope at 200 - 400X magnification. Baculovirus infected cell can be detected by staining with anti-gp64 antibody11.</li>
	<li>Harvest baculovirus supernatant 5 days after transfection and label as the P1 virus stock.</li>
	<li>Store the baculovirus supernatant, which is light sensitive, in the dark with 0.5% bovine serum albumin (BSA) in PBS. Store at 4 &#176;C for the short-term (up to 90 days) or at -80 &#176;C for the long-term.</li>
	<li>Seed 6 &#215; 106 Sf9 cells in a 10-cm tissue culture-treated plate in 10 mL of insect culture medium. Add 1 mL of initial baculovirus supernatant (P1) to the cells.</li>
	<li>Harvest baculovirus supernatant (P2) at 5th day post-infection.</li>
	<li>Seed 50 mL of Sf9 cells (3 x 106 cells/mL) in suspension culture in insect culture medium in a 250-mL polycarbonate Erlenmeyer flask and place the flask in an orbital shaker incubator. Add 2 mL of P2 stock to the cells and shake at 135 rpm while maintaining a constant temperature of 28 &#176;C. 
	Note: Three or four days after infection, the entire Sf9 cell population will show cytopathic effect, which is an indication of high-titer baculovirus production.</li>
	<li>Sequentially amplify the baculovirus supernatants until the desired volume is obtained (P3, P4, &#8230;.) by increasing 10 to 50-fold volume of cell culture in each subsequent infection. 
	Note: P3 is the 3rd round of infection in which 500 mL to 1 L of Sf9 cell culture can be infected with 10 to 20 mL of P2 baculovirus supernatant; P4 is the 4th round of infection in which 5 to 10 L of Sf9 cell culture can be infected with 100 to 200 mL of P3 baculovirus supernatant, and so on. Typically, Sf9 cells are seeded at 3 &#215; 106 cells/mL in serum-free insect culture media.</li>
	<li>Centrifuge the baculovirus supernatant at 1,000 x g for 30 min at 4 &#176;C to remove&#160;cellular debris and treat the baculovirus supernatant with 50 units/mL nuclease (250 units/&#181;L of stock) for 2 h at 37 &#176;C to digest the genomic DNA and RNA. Filter the supernatants through a 0.45 &#956;m filtration unit.</li>
</ol>
3. Preparation of the Chromatography System
<ol>
	<li>Prepare the chromatography system by sequentially rinsing the sample and buffer lines each with sterile water, 1 N sodium hydroxide, water, and wash buffer (20 mM sodium phosphate buffer containing 150 mM sodium chloride, pH 7.0) at a linear flow rate of 50 mL/min.</li>
	<li>Prepare a heparin 50 &#181;m column (10 &#215; 10 cm, 7.9 mL of column volume (CV)) by sequentially running column cleaning buffer (5 CV sterile 20 mM sodium phosphate buffer containing 2 M sodium chloride, pH 7.0) and 5 CV wash buffer at a linear flow rate of 7 mL/min.</li>
</ol>
4. Purification of Baculovirus Vector
<ol>
	<li>Set up the chromatography system as follows:
	<ol>
		<li>Use the sample loading inlet port for loading the baculovirus supernatant.</li>
		<li>Use the buffer inlet port A1 for loading the wash buffer. Prepare a bottle with at least 500 mL of wash buffer and place the wash buffer line into the bottle.</li>
		<li>Use the buffer inlet port A2 for loading the elution buffer (20 mM sodium phosphate with 1.5 M sodium chloride, pH 8.0). Prepare a bottle with at least 500 mL of elution buffer and place the elution buffer line into the bottle.</li>
		<li>Insert several 50 mL conical tubes into the fraction collector to collect the column pass-through baculovirus supernatant, the wash buffer, and the eluted baculovirus.</li>
	</ol>
	</li>
	<li>Equilibrate the heparin column with five 7.9 mL column volumes (CVs) of wash buffer (40 mL) at a linear flow rate of 7.0 mL/min.</li>
	<li>Load 250 mL of baculovirus supernatant onto the heparin column using the sample pump (inlet sample port) of the chromatography system at a linear flow rate of 2.0 mL/min.</li>
	<li>Run 10 CVs (80 mL) of wash buffer through heparin column at a linear flow rate of 2.0 mL/min until the ultraviolet (UV) absorbance curve (280 nm) has returned to baseline and becomes stable.</li>
	<li>Elute the baculovirus particles from the 7.9 mL heparin column with 5 CVs (40 mL) of elution buffer at a linear flow rate of 4.0 mL/min. Watch for a sharp elution peak of protein on the chromatogram when the baculovirus particles dissociate from the heparin column.</li>
	<li>Post-elution, immediately dilute the eluted baculovirus supernatant 10-fold using 20 mM sodium phosphate buffer in water to prevent inactivation of baculovirus particles from osmotic shock during subsequent centrifugation.</li>
	<li>Store 100 &#181;L of each of the fractions, including the column flow-through collected during loading of the baculovirus supernatant, the wash buffer, and the eluate. Infect these baculovirus fractions to HT1080 to evaluate the purification process (See section 6).</li>
	<li>Clean the column with&#160;column cleaning buffer and&#160;wash buffer. Finally, rinse the column with 5 CV&#160;sterile 20% ethanol in water at a linear flow rate of 7.0 mL/min and store at 4 &#176;C.</li>
	<li>Clean the chromatography system with water, 1 N sodium hydroxide, again with water, and&#160;20% ethanol in water at a linear flow rate of 50.0 mL/min and store the system in 20% ethanol.</li>
</ol>
5. Concentration of Baculovirus
<ol>
	<li>Pre-sterilize centrifuge tubes using an autoclave. Add an equal volume of baculovirus supernatant to each ultracentrifuge tubes in a tissue culture hood. Measure the tube weight by a balance and adjust the weight of the contents of the ultracentrifuge tubes with sterile PBS.</li>
	<li>Place each of the ultracentrifuge tubes in opposing bucket of the SW 32 Ti rotor.</li>
	<li>Run the ultracentrifuge at 80,000 &#215; g for 90 min at 4 &#176;C.</li>
	<li>Open the ultracentrifuge tubes in a tissue culture hood and aseptically aspirate the liquid without removing the baculovirus pellet.</li>
	<li>Resuspend the pellet of each tube by vigorously pipetting up and down with 200 &#181;L PBS containing 0.5% (w/vol) BSA.</li>
	<li>Transfer the concentrated baculovirus to cryovials (50 to 100 &#181;L per vial) and store at -80 &#176;C.</li>
</ol>
6. Titration of Baculovirus
<ol>
	<li>The day before infection, count HT1080 cells using a hemocytometer after staining cells with Trypan blue. Seed 1 x 105 HT1080 cells per well in a 6-well plate in 2 mL of Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM) containing 10% fetal bovine serum (FBS). Seed several additional wells to be able to determine the exact number of cells present at the time of infection. Culture the cells in an incubator at 37 &#176;C and 5% CO2. 
	Note: HT1080 cells are used for titration as they express a higher level of heparan sulfate as compared to the other cell lines16. Since HT1080 cells are adherent, titration is simpler and less time consuming as compared to using the traditional Sf9-based titration.</li>
	<li>On the day of infection, determine the number of cells per well by counting cells from wells. Infect the cells by adding the diluted original baculovirus which had been set aside prior to column purification, and with samples from the column flow-through, wash, and elution fractions. For each sample, determine the dilution and volume empirically based on infectious baculovirus particles, and infect each well in triplicate.</li>
	<li>Two days after infection, analyze the cells for the expression of the gene of interest. Cells can be analyzed using a flow cytometer if they express a fluorescent protein (e.g. GFP)17.</li>
	<li>Calculate the titers (infectious unit per mL) based on the number of cells present at the time of infection, the dilution factor, and percentages of fluorescent protein in cells using the formula: (Number of cells during infection &#215; percentage of fluorescent protein positive cells &#215; dilution factor)/volume of baculovirus in mL. 
	Note: For example, if total number of cells per well at the time of infection is 1.2 &#215; 105, the percentage of fluorescent protein is 5%, the dilution factor is 100, and the volume of diluted baculovirus added is 10 &#181;L, the titer is 6 &#215; 107 infectious units (IU)/mL.</li>
</ol>

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The authors declare no conflict of interest.

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">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</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 ...

production-purification-baculovirus-for-gene-therapy

Research

JoVE Journal

Genetics

14.6K Views.  Cincinnati Children's Hospital Medical Center. 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.

Video: Production and Purification of Baculovirus for Gene Therapy Application

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 ...

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, ...

Assessing Stem Cell DNA Integrity for Cardiac Cell Therapy

Stem and stem-cell-derived cells have immense potential as a regenerative therapy for various degenerative diseases. DNA is the storehouse of genetic data in all cells, including stem cells, and its integrity is fundamental to its regenerative ability. Stem cells undergo rapid propagation in labs to achieve the necessary numbers for transplantation. Accelerated cell growth leads to the loss of DNA integrity by accumulated metabolites, such as reactive oxygen, carbonyl, and alkylating agents. Transplanting these cells would result in poor engraftment and regeneration of the deteriorating organ. Moreover, transplanting DNA-damaged cells leads to mutations, DNA instability, cellular senescence, and possibly, life-threatening diseases such as cancer. Therefore, there is an immediate need for a quality control method to evaluate the cell's suitability for transplantation. Here, we provide step-by-step protocols for the assessment of the DNA integrity of stem cells prior to cell transplantation.

Here, we provide a detailed description of an experimental setup for an analysis of the assessment of DNA integrity in stem cells prior to cell transplantation.

Stem and stem-cell-derived cells have immense potential as a regenerative therapy for various degenerative diseases. DNA is the storehouse of genetic data ...

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. ...