Name: electroporation-mediated delivery of cas9 ribonucleoproteins and mrna into freshly isolated primary mouse hepatocytes
Uploaded: 2022-06-02T13:10:00
Description: Watch this Scientific Journal Video about Electroporation-Mediated Delivery of Cas9 Ribonucleoproteins and mRNA into Freshly Isolated Primary Mouse Hepatocytes at JoVE.com

RNC received funding from South Carolina Bioengineering Center of Regeneration and Formation of Tissues Pilot grant supported by the National Institute of General Medical Sciences (NIGMS) of the National Institutes of Health, the American Association for the Study of Liver Diseases Foundation, and American Society of Gene &#38; Cell Therapy under grant numbers P30 GM131959, 2021000920, and 2022000099, respectively. The content is solely the responsibility of the authors and does not necessarily represent the official views of the American Society of Gene &#38; Cell Therapy or the American Association for the Study of Liver Diseases Foundation. The schematic for Figure 1 was created with BioRender.com.

The animal experiments were all performed in compliance with the Institutional Animal Care and Use Committee guidelines and approved protocols at Clemson University. Surgical procedures were performed in anesthetized wild-type C57BL/6J mice between 8 and 10 weeks old.
1. Animal surgery
<ol>
	<li>Preparation of solutions and instruments 
	NOTE: Perfusion solutions and anesthesia cocktail recipes are shown in the Table of Materials.
	<ol>
		<li>Prepare Pe...

<ol>
	<li>Pampols, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inherited+metabolic+rare+disease.">Inherited metabolic rare disease.</a> Advances in Experimental Medicine and Biology. 686, 397-431 (2010).</li><li>Saudubray, J. M., Sedel, F., Walter, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+approach+to+treatable+inborn+metabolic+diseases:+an+introduction.">Clinical approach to treatable inborn metabolic diseases: an introduction.</a> Journal of Inherited Metabolic Disease. 29 (2-3), 261-274 (2006).</li><li>Arnon, R., 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=Liver+transplantation+in+children+with+metabolic+diseases:+the+studies+of+pediatric+liver+transplantation+experience.">Liver transplantation in children with metabolic diseases: the studies of pediatric liver transplantation experience.</a> Pediatric Transplantation. 14 (6), 796-805 (2010).</li><li>Maiorana, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preemptive+liver+transplantation+in+a+child+with+familial+hypercholesterolemia.">Preemptive liver transplantation in a child with familial hypercholesterolemia.</a> Pediatric Transplantation. 15 (2), 25-29 (2011).</li><li>. OPTN/SRTR 2019 Annual Data Report: Liver Available from: <a target="_blank" href="https://srtr.transplant.hrsa.gov/annual_reports/2019/Liver.aspx">https://srtr.transplant.hrsa.gov/annual_reports/2019/Liver.aspx</a> (2019)</li><li>Fabris, L., Strazzabosco, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rare+and+undiagnosed+liver+diseases:+challenges+and+opportunities.">Rare and undiagnosed liver diseases: challenges and opportunities.</a> Translational Gastroenterology and Hepatology. 6, (2020).</li><li>Haugabook, S. J., Ferrer, M., Ottinger, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+and+in+vivo+translational+models+for+rare+liver+diseases.">In vitro and in vivo translational models for rare liver diseases.</a> Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease. 1865 (5), 1003-1018 (2019).</li><li>Xue, 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=Embryonic+lethality+and+vascular+defects+in+mice+lacking+the+notch+ligand+Jagged1.">Embryonic lethality and vascular defects in mice lacking the notch ligand Jagged1.</a> Human Molecular Genetics. 8 (5), 723-730 (1999).</li><li>Lima, A., Maddalo, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SEMMs:+Somatically+engineered+mouse+models.+a+new+tool+for+in+vivo+disease+modeling+for+basic+and+translational+research.">SEMMs: Somatically engineered mouse models. a new tool for in vivo disease modeling for basic and translational research.</a> Frontiers in Oncology. 11, 1117 (2021).</li><li>Kim, S., Kim, D., Cho, S. W., Kim, J., Kim, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+efficient+RNA-guided+genome+editing+in+human+cells+via+delivery+of+purified+Cas9+ribonucleoproteins.">Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins.</a> Genome Research. 24 (6), 1012-1019 (2014).</li><li>Ye, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Seamless+modification+of+wild-type+induced+pluripotent+stem+cells+to+the+natural+CCR5Delta32+mutation+confers+resistance+to+HIV+infection.">Seamless modification of wild-type induced pluripotent stem cells to the natural CCR5Delta32 mutation confers resistance to HIV infection.</a> Proceedings of the National Academy of Sciences. 111 (26), 9591-9596 (2014).</li><li>Straub, C., Granger, A. J., Saulnier, J. L., Sabatini, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPR/Cas9-mediated+gene+knock-down+in+post-mitotic+neurons.">CRISPR/Cas9-mediated gene knock-down in post-mitotic neurons.</a> PLoS One. 9 (8), 105584 (2014).</li><li>Hoban, M. D., 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=CRISPR/Cas9-mediated+correction+of+the+sickle+mutation+in+human+CD34++cells.">CRISPR/Cas9-mediated correction of the sickle mutation in human CD34+ cells.</a> Molecular Therapy. 24 (9), 1561-1569 (2016).</li><li>Rathbone, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electroporation-mediated+delivery+of+Cas9+ribonucleoproteins+results+in+high+levels+of+gene+editing+in+primary+hepatocytes.">Electroporation-mediated delivery of Cas9 ribonucleoproteins results in high levels of gene editing in primary hepatocytes.</a> The CRISPR Journal. , (2022).</li><li>. Benchling [Biology Software] Available from: <a target="_blank" href="https://benchling.com">https://benchling.com</a> (2022)</li><li>Brinkman, E. K., Chen, T., Amendola, M., van Steensel, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Easy+quantitative+assessment+of+genome+editing+by+sequence+trace+decomposition.">Easy quantitative assessment of genome editing by sequence trace decomposition.</a> Nucleic Acids Research. 42 (22), 168 (2014).</li><li>Cabral, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+hepatocytes+and+sinusoidal+endothelial+cells+from+mouse+liver+perfusion.">Purification of hepatocytes and sinusoidal endothelial cells from mouse liver perfusion.</a> Journal of Visualized Experiments: JoVE. (132), e56993 (2018).</li><li>Huang, P., 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=Induction+of+functional+hepatocyte-like+cells+from+mouse+fibroblasts+by+defined+factors.">Induction of functional hepatocyte-like cells from mouse fibroblasts by defined factors.</a> Nature. 475 (7356), 386-389 (2011).</li><li>Severgnini, 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=A+rapid+two-step+method+for+isolation+of+functional+primary+mouse+hepatocytes:+cell+characterization+and+asialoglycoprotein+receptor+based+assay+development.">A rapid two-step method for isolation of functional primary mouse hepatocytes: cell characterization and asialoglycoprotein receptor based assay development.</a> Cytotechnology. 64 (2), 187-195 (2012).</li><li>Jung, Y., Zhao, M., Svensson, 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=Isolation,+culture,+and+functional+analysis+of+hepatocytes+from+mice+with+fatty+liver+disease.">Isolation, culture, and functional analysis of hepatocytes from mice with fatty liver disease.</a> STAR Protocols. 1 (3), 100222 (2020).</li><li>Kim, 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=Three-dimensional+(3D)+printing+of+mouse+primary+hepatocytes+to+generate+3D+hepatic+structure.">Three-dimensional (3D) printing of mouse primary hepatocytes to generate 3D hepatic structure.</a> Annals of Surgical Treatment and Research. 92 (2), 67-72 (2017).</li><li>Li, W. C., Ralphs, K. L., Tosh, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+adult+mouse+hepatocytes.">Isolation and culture of adult mouse hepatocytes.</a> Methods in Molecular Biology. 633, 185-196 (2010).</li><li>Lattanzi, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+CRISPR/Cas9+delivery+to+human+hematopoietic+stem+and+progenitor+cells+for+therapeutic+genomic+rearrangements.">Optimization of CRISPR/Cas9 delivery to human hematopoietic stem and progenitor cells for therapeutic genomic rearrangements.</a> Molecular Therapy. 27 (1), 137-150 (2019).</li><li>Dever, D. P., 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=CRISPR/Cas9+beta-globin+gene+targeting+in+human+haematopoietic+stem+cells.">CRISPR/Cas9 beta-globin gene targeting in human haematopoietic stem cells.</a> Nature. 539 (7629), 384-389 (2016).</li><li>Grompe, M., Tanguay, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Hereditary Tyrosinemia: Pathogenesis, Screening and Management. , 215-230 (2017).</li></ol>

The steps outlined in the protocol for hepatocyte isolation are challenging and require practice for proficiency. There are several key steps for the successful hepatocyte isolation from the liver. First, proper cannulation of the inferior vena cava is essential for complete liver perfusion. The absence of blanching in the liver after perfusion indicates displacement of the catheter (Table 1). The inferior vena cava (retrograde perfusion) was cannulated in the procedure because it is simpler and more acc...

IMDs of the liver are genetic disorders characterized by the deficiency of a crucial hepatic enzyme involved in metabolism that leads to the accumulation of toxic metabolites. Without treatment, IMDs of the liver result in organ failure or premature death1,2. The only curative option for patients with IMDs of the liver is orthotopic liver transplantation, which is limited due to the low availability of donor organs and complications from immunosuppressive therapy following the procedure3,4. According to recent data collected by the Organ Procurement and Transplantation Network, only 40%-46% of adult patients on the liver transplant waiting list receive an organ, while 12.3% of these patients die while on the waiting list5. Moreover, only 5% of all the rare liver diseases have an FDA-approved treatment6. It is clear that there is a critical need for novel treatments for IMDs of the liver. However, appropriate disease models are required to develop new therapeutic options.
Modeling human diseases using in vitro and in vivo systems remains an obstacle for developing effective therapies and studying the pathology of IMDs of the liver. Hepatocytes from patients with rare liver diseases are challenging to obtain7. Animal models are crucial for developing an understanding of disease pathology and for testing therapeutic strategies. However, one obstacle is generating models from embryos carrying lethal mutations. For example, attempts to create mouse models of Alagille Syndrome (ALGS) with embryos containing homozygous deletions of a 5 kb sequence near the 5&#8242;-end of the Jag1 gene resulted in the early death of the embryos8. In addition, generating mouse models by gene editing in embryonic stem cells can be time- and resource-intensive9. Lastly, mutations will appear outside the targeted tissue, leading to confounding variables that may impede study of the disease9. Somatic gene editing would allow for easier editing in liver tissue and bypasses the challenges associated with generating models using embryonic stem cells.
Electroporation enables the delivery of CRISPR-Cas9 directly into the nucleus by applying high-voltage currents to permeabilize the cell membrane and is compatible with many cell types, including those that are intransigent to transfection techniques, such as human embryonic stem cells, pluripotent stem cells, and neurons10,11,12. However, low viability is a potential drawback of electroporation; optimizing the procedure can yield high levels of delivery while limiting toxicity13. A recent study demonstrates the feasibility of electroporating CRISPR-Cas9 components into primary mouse and human hepatocytes as a highly efficient approach14. Ex vivo electroporation in hepatocytes has the potential to be applied to generate new mouse models for human IMDs of the liver.
This protocol provides a detailed step-by-step procedure for isolating mouse hepatocytes from the liver and subsequently electroporating CRISPR-Cas9 as RNP complexes, consisting of Cas9 protein and synthetic single-guide RNA (sgRNA), or Cas9 mRNA combined with sgRNA to obtain high levels of on-target gene editing. In addition, the protocol provides methods for quantifying gene editing efficiency, viability, and functionality following electroporation of CRISPR-Cas9 into freshly isolated mouse hepatocytes.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>0.2 mL PCR 8-tube FLEX-FREE strip, attached clear flat caps, natural</td>
			<td>USA Scientific</td>
			<td>1402-4700</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well Collagen Plates</td>
			<td>Advanced Biomatrix</td>
			<td>5073</td>
			<td></td>
		</tr>
		<tr>
			<td>accuSpin Micro 17R</td>
			<td>Fisher Scientific</td>
			<td>13-100-675</td>
			<td></td>
		</tr>
		<tr>
			<td>All-in-one Fluorescent Microscope</td>
			<td>Keyence</td>
			<td>BZ-X810</td>
			<td></td>
		</tr>
		<tr>
			<td>Analog Vortex Mixer</td>
			<td>VWR</td>
			<td>97043-562</td>
			<td></td>
		</tr>
		<tr>
			<td>ART Wide BORE filtered tips 1,000 &#181;L</td>
			<td>ThermoFisher Scientific</td>
			<td>2079GPK</td>
			<td></td>
		</tr>
		<tr>
			<td>Automated Cell Counter</td>
			<td>Bio-Rad Laboratories</td>
			<td>TC20</td>
			<td></td>
		</tr>
		<tr>
			<td>Blue Wax Dissection Tray</td>
			<td>Braintree Scientific Inc.</td>
			<td>DTW1</td>
			<td>9&#34; x 6.5&#34; x 1/2&#34;+F21</td>
		</tr>
		<tr>
			<td>Cell scraper/lifter</td>
			<td>Argos Technologies</td>
			<td>UX-04396-53</td>
			<td>Non-pyrogenic, sterile</td>
		</tr>
		<tr>
			<td>Conical tubes (15 mL)</td>
			<td>Fisher Scientific</td>
			<td>339650</td>
			<td></td>
		</tr>
		<tr>
			<td>Conical tubes (50 mL)</td>
			<td>Fisher Scientific</td>
			<td>14-432-22</td>
			<td></td>
		</tr>
		<tr>
			<td>Cotton applicators</td>
			<td>Fisher Scientific</td>
			<td>22-363-170</td>
			<td></td>
		</tr>
		<tr>
			<td>Curved scissors</td>
			<td>Cooper Surgical</td>
			<td>62131</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable Petri Dishes</td>
			<td>Falcon</td>
			<td>351029</td>
			<td>100mm,sterile</td>
		</tr>
		<tr>
			<td>Disposable Petri Dishes</td>
			<td>VWR</td>
			<td>25373-100</td>
			<td>35mm, sterile</td>
		</tr>
		<tr>
			<td>Epoch Microplate Spectrophotometer</td>
			<td>BioTek Instruments</td>
			<td>250082</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon Cell strainer (70 &#181;m)</td>
			<td>Fisher Scientific</td>
			<td>08-771-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Cooper Surgical</td>
			<td>61864</td>
			<td>Euro-Med Adson Tissue Forceps</td>
		</tr>
		<tr>
			<td>IV catheters&#160;</td>
			<td>BD</td>
			<td>382612</td>
			<td>24 G x 0.75 in</td>
		</tr>
		<tr>
			<td>IV infusion set</td>
			<td>Baxter</td>
			<td>2C6401</td>
			<td></td>
		</tr>
		<tr>
			<td>MyFuge 12 Mini Centrifuge</td>
			<td>Benchmark Scientific</td>
			<td>1220P38</td>
			<td></td>
		</tr>
		<tr>
			<td>Needles</td>
			<td>Fisher Scientific</td>
			<td>05-561-20</td>
			<td>25 G</td>
		</tr>
		<tr>
			<td>Nucleofector 2b Device</td>
			<td>Lonza</td>
			<td>AAB-1001</td>
			<td>Program T-028 was used for electroporation in mouse hepatocytes</td>
		</tr>
		<tr>
			<td>Peristaltic Pump</td>
			<td>Masterflex</td>
			<td>&#160;HV-77120-42</td>
			<td>10 to 60 rpm; 90 to 260 VAC</td>
		</tr>
		<tr>
			<td>Precision pump tubing</td>
			<td>Masterflex</td>
			<td>HV-96410-14</td>
			<td>25 ft, silicone</td>
		</tr>
		<tr>
			<td>Primaria Culture Plates</td>
			<td>Corning Life Sciences</td>
			<td>353846</td>
			<td>Nitrogen-containing tissue culture plates</td>
		</tr>
		<tr>
			<td>Serological Pipets (25 mL)</td>
			<td>Fisher Scientific</td>
			<td>12-567-604</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringes</td>
			<td>BD</td>
			<td>329464</td>
			<td>1 mL, sterile</td>
		</tr>
		<tr>
			<td>T100 Thermal Cycler</td>
			<td>Bio-Rad Laboratories</td>
			<td>1861096</td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath</td>
			<td>ALT</td>
			<td>27577</td>
			<td>Thermo Scientific Precision Microprocessor Controlled 280 Series, 2.5 L</td>
		</tr>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Alt-R S.p. Cas9 Nuclease V3</td>
			<td>IDT</td>
			<td>1081058</td>
			<td></td>
		</tr>
		<tr>
			<td>Beckman Coulter AMPure XP, 5 mL</td>
			<td>Fisher Scientific</td>
			<td>NC9959336</td>
			<td></td>
		</tr>
		<tr>
			<td>CleanCap Cas9 mRNA</td>
			<td>Trilink Biotechnologies</td>
			<td>L-7606-100</td>
			<td></td>
		</tr>
		<tr>
			<td>CleanCap&#160;EGFP mRNA</td>
			<td>Trilink Biotechnologies</td>
			<td>L-7201-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning Matrigel Matrix</td>
			<td>Corning Life Sciences</td>
			<td>356234</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>ThermoFisher Scientific</td>
			<td>11885076</td>
			<td>Low glucose, pyruvate</td>
		</tr>
		<tr>
			<td>Ethanol 70%</td>
			<td>VWR</td>
			<td>71001-652</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Thermoscientific</td>
			<td>26140-079</td>
			<td></td>
		</tr>
		<tr>
			<td>Hepatocyte Maintenance Medium (MM)</td>
			<td>Lonza</td>
			<td>MM250</td>
			<td></td>
		</tr>
		<tr>
			<td>Hepatocyte Plating Medium (PM)</td>
			<td>Lonza</td>
			<td>MP100</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse Albumin ELISA Kit</td>
			<td>Fisher Scientific</td>
			<td>NC0010653</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse/Rat Hepatocyte Nucleofector Kit</td>
			<td>Lonza</td>
			<td>VPL-1004</td>
			<td></td>
		</tr>
		<tr>
			<td>OneTaq HotStart DNA Polymerase</td>
			<td>New England Biolabs</td>
			<td>M0481L</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS 10x pH 7.4&#160;</td>
			<td>Thermoscientific</td>
			<td>70011-044</td>
			<td>No calcium or magnesium chloride</td>
		</tr>
		<tr>
			<td>Percoll (PVP solution)</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-500790A</td>
			<td></td>
		</tr>
		<tr>
			<td>Periodic acid</td>
			<td>Sigma-Aldrich</td>
			<td>P7875-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Permount Mounting Medium</td>
			<td>VWR</td>
			<td>100496-550</td>
			<td></td>
		</tr>
		<tr>
			<td>QuickExtract DNA Extraction Solution</td>
			<td>Lucigen Corporation</td>
			<td>QE05090</td>
			<td></td>
		</tr>
		<tr>
			<td>Schiff&#8217;s fuchsin-sulfite reagent</td>
			<td>Sigma-Aldrich</td>
			<td>S5133</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.25%)</td>
			<td>ThermoFisher Scientific</td>
			<td>25200056</td>
			<td>Phenol red</td>
		</tr>
		<tr>
			<td>Vybrant MTT Cell Viability Assay</td>
			<td>ThermoFisher Scientific</td>
			<td>V13154</td>
			<td></td>
		</tr>
		<tr>
			<td>Perfusion Solution 1 (pH 7.4, filter sterilized)&#160;</td>
			<td></td>
			<td></td>
			<td>Stable at 4 &#176;C for 2 months</td>
		</tr>
		<tr>
			<td>EBSS</td>
			<td>Fisher Scientific</td>
			<td>14155063</td>
			<td>Complete to 200 mL</td>
		</tr>
		<tr>
			<td>EGTA (0.5 M)</td>
			<td>Bioworld</td>
			<td>40520008-1</td>
			<td>200 &#181;L</td>
		</tr>
		<tr>
			<td>HEPES (pH 7.3, 1 M)</td>
			<td>ThermoFisher Scientific</td>
			<td>AAJ16924AE</td>
			<td>2 mL&#160;</td>
		</tr>
		<tr>
			<td>Perfusion Solution 2 (pH 7.4, filter sterilized)&#160;</td>
			<td></td>
			<td></td>
			<td>Stable at 4 &#176;C for 2 months</td>
		</tr>
		<tr>
			<td>CaCl2&#183;2H20 (1.8 mM)</td>
			<td>Sigma</td>
			<td>C7902-500G</td>
			<td>360 &#181;L of 1 M stock&#160;</td>
		</tr>
		<tr>
			<td>EBSS (no calcium, no magnesium, no phenol red)</td>
			<td>Fisher Scientific</td>
			<td>14-155-063</td>
			<td>Complete to 200 mL</td>
		</tr>
		<tr>
			<td>HEPES (1 M)</td>
			<td>ThermoFisher Scientific</td>
			<td>AAJ16924AE</td>
			<td>2 mL&#160;</td>
		</tr>
		<tr>
			<td>MgSO4&#183;7H20 (0.8 mM)</td>
			<td>Sigma</td>
			<td>30391-25G</td>
			<td>160 &#181;L of 1 M stock</td>
		</tr>
		<tr>
			<td>Perfusion Solution 3&#160;</td>
			<td></td>
			<td></td>
			<td>Prepared fresh prior to use</td>
		</tr>
		<tr>
			<td>Solution 2</td>
			<td></td>
			<td></td>
			<td>50 mL</td>
		</tr>
		<tr>
			<td>Liberase</td>
			<td>Roche</td>
			<td>5401127001</td>
			<td>0.094 Wunsch units/mL</td>
		</tr>
		<tr>
			<td>Mouse Anesthetic Cocktail&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Acepromzine</td>
			<td></td>
			<td></td>
			<td>0.25 mg/mL final concentration</td>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td></td>
			<td></td>
			<td>7.5 mg/mL final concentration</td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td></td>
			<td></td>
			<td>1.5 mg/mL final concentration</td>
		</tr>
		<tr>
			<td>Software</td>
			<td>URL</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Benchling</td>
			<td>https://www.benchling.com/</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>https://imagej.nih.gov/ij/</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TIDE: Tracking of Indels by Decomposition</td>
			<td>https://tide.nki.nl/</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

electroporation-mediated delivery of cas9 ribonucleoproteins and mrna into freshly isolated primary mouse hepatocytes

This protocol describes a fast and effective method for isolating primary mouse hepatocytes followed by electroporation-mediated delivery of CRISPR-Cas9 as ribonucleoproteins (RNPs) and mRNA. Primary mouse hepatocytes were isolated using a three-step retrograde perfusion method resulting in high yields of up to 50 &#215; 106 cells per liver and cell viability of &#62;85%. This protocol provides detailed instructions for plating, staining, and culturing hepatocytes. The results indicate that electroporation provides a high transfection efficiency of 89%, as measured by the percentage of green fluorescent protein (GFP)-positive cells and modest cell viability of &#62;35% in mouse hepatocytes.
To demonstrate the utility of this approach, CRISPR-Cas9 targeting the hydroxyphenylpyruvate dioxygenase gene was electroporated into primary mouse hepatocytes as proof-of-principle gene editing to disrupt a therapeutic gene related to an inherited metabolic disease (IMD) of the liver. A higher on-target edit of 78% was observed for RNPs compared to 47% editing efficiency with mRNA. The functionality of hepatocytes was evaluated in vitro using an albumin assay that indicated that delivering CRISPR-Cas9 as RNPs and mRNA results in comparable cell viability in primary mouse hepatocytes. A promising application for this protocol is the generation of mouse models for human genetic diseases affecting the liver.

Isolation of plateable primary hepatocytes from the liver 
The overall process of liver perfusion and hepatocyte isolation is illustrated in Figure 1. In this experiment, wild-type, 8-10-week-old C57BL6/6J mice were used. The procedure is expected to yield 20-50 &#215; 106 cells per mouse with a viability between 85% and 95%. If the viability is &#60;70%, percoll treatment should be followed to remove dead cells. Freshly isolated hepatocytes should be plated ...

Watch this Scientific Journal Video about Electroporation-Mediated Delivery of Cas9 Ribonucleoproteins and mRNA into Freshly Isolated Primary Mouse Hepatocytes at JoVE.com

Electroporation-Mediated Delivery of Cas9 Ribonucleoproteins and mRNA into Freshly Isolated Primary Mouse Hepatocytes

This protocol describes techniques for isolating primary mouse hepatocytes from the liver and electroporating CRISPR-Cas9 as ribonucleoproteins and mRNA to disrupt a therapeutic target gene associated with an inherited metabolic disease of the liver. The methods described result in high viability and high levels of gene modification after electroporation.

This protocol describes a fast and effective method for isolating primary mouse hepatocytes followed by electroporation-mediated delivery of CRISPR-Cas9 ...

The main advantage of our technique is that it is an easy, fast, and reproducible approach for editing genes in primary mouse hepatocytes as a way to generate in vitro and in vivo disease models. Using our technique, it may be possible to knock down a target gene in isolated hepatocytes and then transplant them back into the patient, to replace diseased cells with gene edited hepatocytes Inexperienced researchers might struggle with cannulation and keeping the catheter steady throughout the perfusion. The users are advised to carefully read and practice the procedure and the troubleshooting methods before getting started.Demonstrating the liver perfusion and hepatocyte isolation will be Tina Parker a clinical facility manager and Ilayda Ates, a graduate research assistant. The electroporation procedure will be demonstrated by Callie Stuart, a graduate research assistant from my laboratory. Under a surgical plane of anesthesia.Begin the liver perfusion by making a U-shaped lateral incision in the abdomen skin using scissors, and expand the hole through the sides. Continue opening the skin to the rib cage. Using scissors, cut through the peritoneum to expose the abdominal cavity up to the ribcage.Being careful not to nick any organs. Move the intestines to the right side using the back of forceps or the blunt surface of scissors. Identify the portal vein and the inferior vena cava.Start the pump to flush pre-warmed perfusion solution through the catheter. Stop the pump and remove the catheter. Then insert the tip of the needle connected to the catheter into the inferior vena cava at a 10 to 20 degree angle to the vein, remove the needle from the catheter.Then gently push the catheter into the vein in a movement parallel to the vein. Connect the tubing to the catheter without moving the catheter further into the vein. Start the pump with a flow rate of two milliliters per minute.Look for the liver immediately turning pale as a sign that the perfusion is successful. Cut the portal vein using scissors. Gradually increase the flow rate to five milliliters per minute.Once the perfusion solution three is flowing through carefully monitor the elasticity of the liver. To determine if the liver has softened, gently press the liver with a cotton swab or forceps and check whether indentations form on the liver. Be careful not to over perfuse to avoid loss of cell viability.Once the liver softens after 30 to 50 milliliters of perfusion solution three has flowed through, stop the pump, remove the catheter and the gallbladder. Careful not to spill its contents. Then carefully dissect the liver.Place the liver into a 100 millimeter Petri dish containing ice cold DME M with 10%FBS medium. Swirl the Petri dish gently to remove possible blood clots. Transfer the liver to a new Petri dish containing ice cold DME M and 10%FBS medium.To release cells from the liver capsule, place the Petri dish on ice then using two pairs of sterile forceps, gently tear apart the liver capsule on all lobes. Gently swirl the liver around in the medium using a cell lifter to release the hepatocytes. Continue this motion until the liver becomes very small and the suspension turns brown and opaque.Remove the liver tissue remains from the plate and using a 25 milliliter serological pipette set to low speed, carefully transfer the cell suspension to a 50 milliliter conical tube pre chilled on ice and fitted with a 100 micrometer cell strainer. Add fresh ice cold DMEM, and 10%FBS medium to the Petri dish to wash out and collect the remaining cells from the surface. Then, transfer to the 50 milliliter conical tube.Repeat this step until all the cells are collected. To wash and purify hepatocytes from non parenchymal cells, centrifuge the cells collected in the 50 milliliter conical tube at 50 RCF at four degrees Celsius for five minutes at low deceleration or without any breaks. Discard the super-naton containing debris and non-parenchymal cells.Then re-suspend the pellet in the leftover super-naton by gently swirling the tube. After pellet re-suspension add 30 milliliters of fresh ice cold DMEM and 10%FBS medium. Begin preparing the CRISPR CAS 9 substrates media, cells and the electroporation instrument by turning on the power button on the electroporation device.Then set the electroporation program by pressing the X button and then the down arrow button until the program T 0 28 appears on the screen. Prepared the destination wells for the electroporated hepatocytes by adding 1.5 milliliters of PM to six well collagen one coated plates. Incubate the plates in a 37 degree Celsius incubator until ready to use.In PCR split strip tubes, prepare the CAS 9 RMP and mRNA complexes as described in the text manuscript. Store the mRNA SG RNA mix on ice until electroporation. Separately, prepare a tube containing one microgram of E G F P mRNA to verify successful electroporation using fluorescence microscopy.Centrifuge the electroporation reaction at 100 RCF for two minutes at four degrees Celsius. Remove the supernaton from the cell pellet and add 100 microliters of nucleoffection solution per reaction along the side of the tube wall. Re suspend the hepatocytes in the electroporation solution by rocking the tube gently by hand.Once the hepatocytes are evenly dispersed in the electroporation solution, transfer 100 microliters of the hepatocyte suspension to the strip tubes containing the CAS nine complexes. Transfer the contents of the strip well to an electroporation vessel. Place the nucleo-vet vessel into the slot on the electroporation device.Electroporate the hepatocytes by pressing the X button. After electroporation, wait for a screen to pop up on the device that says:Okay. Press the X button again and remove the vessel.Incubate the electroporated vessel on ice for 15 minutes. Add 500 microliters a prew-armed PM to the electroporation vessel. Transfer 300 microliters of the electroporation reaction to each destination well on the pre-warmed plate.Incubate the plates overnight at 37 degrees Celsius. Add the membrane matrix to ice cold hepatocyte maintenance medium and mix by pipetting up and down 10 times. Remove the medium from the wells designated for gene editing analysis 24 hours after plating the cells.Pipette the overlay mixture slowly on top of the cells and place the plate back at 37 degrees Celsius. At 24 hours after plating, transfer the conditioned medium from the well designated for MTT and albumin assays and store in a micro-fuge tube until ready to perform the albumin assay. Proceed with analysis of on target CAS nine activity by extracting the genomic DNA and setting up the PCR as described in the text manuscript.Within 3 to 12 hours of plating the hepatocytes adhered to the plate and the cell morphology assumed a typical polygonal or hexagonal appearance within 24 hours The purity of the isolated cells was verified via glycogen staining using the periodic acid shift reagent at 24 hours after plating. The cytoplasm of the stained hepatocytes appeared magenta. The hepatocytes were imaged 24 hours after electroporation and on average, 89.8%of the hepatocytes were GFP positive.At three days after electroporation CAS nine induced in the HPD locus where analyzed and the results showed on target indels of 47.4%in hepatocytes electroporated with CRISPR Cas nine mRNA and 78.4%indels for the CAS nine RNP. The MTT assay showed 35.4 and 45.9%viability in hepatocytes treated with CRISPR Cas nine mRNA and R N P.Consistent with the MTT results the normalized albumin levels were 31.8%in hepatocytes treated with CAS nine RNA and 34.5%for CAS nine r p. The most important thing to remember is that proper cannulation affects all of the later steps and the success of the procedure.After this procedure, the hepatocytes can be transplanted and mice to investigate their engraftments. Moreover, the genomic DNA of plated hepatocytes can be extracted to analyze the efficiency and specificity of CAS nine editing.

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