Name: laser-assisted lentiviral gene delivery to mouse fertilized eggs
Uploaded: 2018-11-01T14:30:00
Description: Watch this Scientific Journal Video about Laser-assisted Lentiviral Gene Delivery to Mouse Fertilized Eggs at JoVE.com

This research was supported by the Intramural Research Program of the National Institute of Health (NIH), National Institute of Environmental Health Sciences (NIEHS). We are grateful to Dr. Robert Petrovich and Dr. Jason Williams for critical reading of the manuscript and helpful advice. We would also like to acknowledge and thank Dr. Bernd Gloss, the Knockout core, the Flow Cytometry Facility, the Fluorescence Microscopy and Imaging Center, and the Comparative Medicine Branch facilities of the NIEHS for their technical contributions. We would like to thank Mr. David Goulding from the Comparative Medicine Branch and Ms. Lois Wyrick of the Imaging Center at the NIEHS for providing us with photographs and illustrations.

All animal procedures and treatments used in this protocol were in compliance with the NIH/NIEHS animal care guidelines and were approved by the Animal Care and Use Committee (ACUC) at the NIH/NIEHS, Animal Protocol 2010-0004.
1. Preparations
<ol>
	<li>Prepare/purchase recombinant non-propagating lentiviruses carrying your gene of interest. In this study, lentivirus SBI511 expressing copepod green fluorescent protein (copGFP; abbreviated to GFP) from an elongation factor 1a promoter was used...

<ol>
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G., Taylor, L., Sang, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+conservation+between+rodents+and+chicken+of+regulatory+sequences+driving+skeletal+muscle+gene+expression+in+transgenic+chickens.">Functional conservation between rodents and chicken of regulatory sequences driving skeletal muscle gene expression in transgenic chickens.</a> BMC Developmental Biology. 10, 26 (2010).</li><li>Zhang, 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=Production+of+transgenic+pigs+mediated+by+pseudotyped+lentivirus+and+sperm.">Production of transgenic pigs mediated by pseudotyped lentivirus and sperm.</a> PLoS One. 7 (4), e35335 (2012).</li><li>Zhang, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenic+quail+production+by+microinjection+of+lentiviral+vector+into+the+early+embryo+blood+vessels.">Transgenic quail production by microinjection of lentiviral vector into the early embryo blood vessels.</a> PLoS One. 7 (12), e50817 (2012).</li><li>Wassarman, P. 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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=Production+of+transgenic+rats+by+lentiviral+transduction+of+male+germ-line+stem+cells.">Production of transgenic rats by lentiviral transduction of male germ-line stem cells.</a> Proceedings of National Academy of Science U S A. 99 (23), 14931-14936 (2002).</li><li>Kanatsu-Shinohara, 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=Production+of+transgenic+rats+via+lentiviral+transduction+and+xenogeneic+transplantation+of+spermatogonial+stem+cells.">Production of transgenic rats via lentiviral transduction and xenogeneic transplantation of spermatogonial stem cells.</a> Biology of Reproduction. 79 (6), 1121-1128 (2008).</li><li>Dann, C. T., Garbers, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+knockdown+rats+by+lentiviral+transduction+of+embryos+with+short+hairpin+RNA+transgenes.">Production of knockdown rats by lentiviral transduction of embryos with short hairpin RNA transgenes.</a> Methods in Molecular Biology. 450, 193-209 (2008).</li><li>Chandrashekran, 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=Efficient+generation+of+transgenic+mice+by+lentivirus-mediated+modification+of+spermatozoa.">Efficient generation of transgenic mice by lentivirus-mediated modification of spermatozoa.</a> FASEB Journal. 28 (2), 569-576 (2014).</li><li>Woods, S. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser-assisted+in+vitro+fertilization+facilitates+fertilization+of+vitrified-warmed+C57BL/6+mouse+oocytes+with+fresh+and+frozen-thawed+spermatozoa,+producing+live+pups.">Laser-assisted in vitro fertilization facilitates fertilization of vitrified-warmed C57BL/6 mouse oocytes with fresh and frozen-thawed spermatozoa, producing live pups.</a> PLoS One. 9 (3), e91892 (2014).</li><li>Tanaka, N., Takeuchi, T., Neri, Q. V., Sills, E. S., Palermo, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser-assisted+blastocyst+dissection+and+subsequent+cultivation+of+embryonic+stem+cells+in+a+serum/cell+free+culture+system:+applications+and+preliminary+results+in+a+murine+model.">Laser-assisted blastocyst dissection and subsequent cultivation of embryonic stem cells in a serum/cell free culture system: applications and preliminary results in a murine model.</a> Journal of Translational Medicine. 4, 20 (2006).</li><li>Martin, N. 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=En+masse+lentiviral+gene+delivery+to+mouse+fertilized+eggs+via+laser+perforation+of+zona+pellucida.">En masse lentiviral gene delivery to mouse fertilized eggs via laser perforation of zona pellucida.</a> Transgenic Research. 27 (1), 39-49 (2018).</li><li>Lawitts, J. A., Biggers, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culture+of+preimplantation+embryos.">Culture of preimplantation embryos.</a> Methods in Enzymology. 225, 153-164 (1993).</li><li>Stein, P., Schindler, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+oocyte+microinjection,+maturation+and+ploidy+assessment.">Mouse oocyte microinjection, maturation and ploidy assessment.</a> Journal of Visualized Experiments. (53), (2011).</li><li>Nagy, A., Gertsenstein, M., Vintersten, K., Behringer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caesarean+section+and+fostering.">Caesarean section and fostering.</a> CSH Protocols. 2006 (2), (2006).</li><li>Chum, P. Y., Haimes, J. D., Andre, C. P., Kuusisto, P. K., Kelley, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genotyping+of+plant+and+animal+samples+without+prior+DNA+purification.">Genotyping of plant and animal samples without prior DNA purification.</a> Journal of Visualized Experiments. (67), (2012).</li><li>Bin Ali, 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=Improved+pregnancy+and+birth+rates+with+routine+application+of+nonsurgical+embryo+transfer.">Improved pregnancy and birth rates with routine application of nonsurgical embryo transfer.</a> Transgenic Research. 23 (4), 691-695 (2014).</li><li>Liu, K. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrase-deficient+lentivirus:+opportunities+and+challenges+for+human+gene+therapy.">Integrase-deficient lentivirus: opportunities and challenges for human gene therapy.</a> Current Gene Therapy. 14 (5), 352-364 (2014).</li><li>Sauvain, M. O., 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=Genotypic+features+of+lentivirus+transgenic+mice.">Genotypic features of lentivirus transgenic mice.</a> Journal of Virology. 82 (14), 7111-7119 (2008).</li></ol>

The ability of the lentiviruses to integrate into their host genome makes them an ideal vector for stable gene delivery. Lentiviral vectors can carry up to 8.5 kilobase pair (kbp) of genetic material that can accommodate cell-specific or inducible promoters, selection markers, or fluorescent moieties. Incorporated genomic material can replicate as part of their host genome and be regulated to express or deactivate at desired time points. These vectors allow for spatiotemporal control over gene expression at various stage...

This method provides detailed instructions for permeabilizing the zona pellucida of mouse fertilized eggs to make embryonic cells accessible for gene delivery by lentiviruses. Lentiviruses are designed by nature for efficient gene delivery to mammalian cells. They infect dividing and non-dividing cells and integrate the lentiviral genome into their host chromosomes1. The range of lentiviral host cells is readily expanded by pseudotyping the recombinant lentivirus with the vesicular stomatitis virus glycoprotein (VSV-G), due to the broad tropism of the VSV-G protein2. Following transduction, lentiviral genes are stably integrated and expressed as part of their host chromosomes creating an ideal tool for generating transgenic animals. If delivered to early stage embryonic cells, the lentiviral genome is replicated and expressed in the entire organism. Lentiviral transduction has led to the production of mice, rat, chicken, quail and pig3,4,5,6,7 among other species of transgenics. The typical method of lentiviral gene delivery, however, requires skilled technicians and specialized equipment to overcome the zona pellucida barrier that encapsulates the early stage embryos. The overall goal of this method is to describe how to permeabilize the zona using a laser to facilitate lentiviral gene delivery.
Mammalian eggs are surrounded by the zona pellucida which hardens following fertilization to protect the fertilized eggs against polyspermy and to limit environmental interactions8,9. The zona forms a barrier that keeps lentiviruses away from the embryonic cells until the embryos are hatched as a blastocyst. Cultured mouse fertilized eggs hatch after 4 days and must be implanted into pseudopregnant mice prior to hatching for normal development into pups. Therefore, for transduction, lentiviruses are microinjected before hatching from the zona into the perivitelline cavity, the space between the zona and the embryonic cells.
The zona pellucida is often removed for in vitro fertilization of human eggs to increase the fertilization rate10. However, chemical removal of mouse zona pellucida adversely affects mouse embryo development and is harmful to embryonic cells11,12. Other methods for gene delivery to mouse fertilized eggs overcome the zona pellucida barrier by direct microinjection of DNA into the cell nucleus13. Pronuclear microinjection is an efficient means of delivering genes to embryos. However, since each embryo is held in place individually for microinjection, the practice can be laborious and time consuming for a novice user.
Other methods such as electroporation and photoporation are useful for transient and short-term gene delivery to mouse fertilized eggs14,15,16. These methods are extensively used for delivering CRISPR-Cas9 components and recombinases. However, electroporation and photoporation delivery of genes cannot be used efficiently to create transgenics. Spermatozoa that are collected from punctured mouse epididymis can also be transduced by lentiviruses and used for in vitro fertilization to produce transgenic animals17,18,19,20.
In this protocol, the lentiviral gene delivery to mouse embryos is facilitated by permeabilizing the zona using a laser. The XYClone laser was developed as an aid for in vitro fertilization21 and cultivation of embryonic stem cells22. It is a small apparatus that is simple to setup and easy to use. Once installed on a microscope, it occupies the space of an objective lens and the accompanying software allows for aiming the laser while looking through the microscope eyepieces (see Protocol: section 3). Once the zona is perforated by the XYClone laser, lentiviruses can be introduced into the culture media for gene delivery23. Multiple lentiviruses could be used to simultaneously deliver several genes for chromosomal incorporation.
This protocol will describe how to isolate and culture mouse fertilized eggs, illustrates the use of laser for perforation of the zona pellucida, and demonstrates the transduction of mouse embryonic cells by lentiviruses.

<table>
	<tbody>
		<tr>
			<td>CD510B-1 plasmid</td>
			<td>System Biosciences</td>
			<td>CD510B-1</td>
			<td>used to package the lentivirus expressing EF1a-copGFP</td>
		</tr>
		<tr>
			<td>Dimethylpolysiloxane</td>
			<td>Sigma</td>
			<td>DMPS5X</td>
			<td>culturing embryos</td>
		</tr>
		<tr>
			<td>hyaluronidase</td>
			<td>Sigma</td>
			<td>H3506</td>
			<td>used to remove cumulus cells</td>
		</tr>
		<tr>
			<td>XYClone Laser</td>
			<td>Hamilton Thorne Biosciences</td>
			<td></td>
			<td>perforating mouse fertilized eggs</td>
		</tr>
		<tr>
			<td>Non-Surgical Embryo Transfer (NSET) Device</td>
			<td>ParaTechs</td>
			<td>60010</td>
			<td>NSET of embryos</td>
		</tr>
		<tr>
			<td>KSOM medium</td>
			<td>Millipore</td>
			<td>MR-020P-5F</td>
			<td>culturing embryos</td>
		</tr>
		<tr>
			<td>Composition of KSOM:</td>
			<td></td>
			<td></td>
			<td>mg/100mL</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td></td>
			<td></td>
			<td>555</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td></td>
			<td></td>
			<td>18.5</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td></td>
			<td></td>
			<td>4.75</td>
		</tr>
		<tr>
			<td>MgSO4 7H2O</td>
			<td></td>
			<td></td>
			<td>4.95</td>
		</tr>
		<tr>
			<td>CaCl2 2H2O</td>
			<td></td>
			<td></td>
			<td>25</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td></td>
			<td></td>
			<td>210</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td></td>
			<td></td>
			<td>3.6</td>
		</tr>
		<tr>
			<td>Na-Pyruvate</td>
			<td></td>
			<td></td>
			<td>2.2</td>
		</tr>
		<tr>
			<td>DL-Lactic Acid, sodium salt</td>
			<td></td>
			<td></td>
			<td>0.174mL</td>
		</tr>
		<tr>
			<td>10mM EDTA</td>
			<td></td>
			<td></td>
			<td>100&#181;L</td>
		</tr>
		<tr>
			<td>Streptomycin</td>
			<td></td>
			<td></td>
			<td>5</td>
		</tr>
		<tr>
			<td>Penicillin</td>
			<td></td>
			<td></td>
			<td>6.3</td>
		</tr>
		<tr>
			<td>0.5% phenol red</td>
			<td></td>
			<td></td>
			<td>0.1mL</td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td></td>
			<td></td>
			<td>14.6</td>
		</tr>
		<tr>
			<td>MEM Essential Amino Acids</td>
			<td></td>
			<td></td>
			<td>1mL</td>
		</tr>
		<tr>
			<td>MEM Non-essential AA</td>
			<td></td>
			<td></td>
			<td>0.5mL</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td></td>
			<td></td>
			<td>100</td>
		</tr>
		<tr>
			<td>M2 medium</td>
			<td>Millipore</td>
			<td>MR-015-D</td>
			<td>culturing embryos</td>
		</tr>
		<tr>
			<td>Composition of M2:</td>
			<td></td>
			<td></td>
			<td>mg/100mL</td>
		</tr>
		<tr>
			<td>Calcium Chloride</td>
			<td></td>
			<td></td>
			<td>25.1</td>
		</tr>
		<tr>
			<td>Magnesium Sulfate (anhydrous)</td>
			<td></td>
			<td></td>
			<td>16.5</td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td></td>
			<td></td>
			<td>35.6</td>
		</tr>
		<tr>
			<td>Potassium Phosphate, Monobasic</td>
			<td></td>
			<td></td>
			<td>16.2</td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate</td>
			<td></td>
			<td></td>
			<td>35</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td></td>
			<td></td>
			<td>553.2</td>
		</tr>
		<tr>
			<td>Albumin, Bovine Fraction</td>
			<td></td>
			<td></td>
			<td>400</td>
		</tr>
		<tr>
			<td>D-Glucose</td>
			<td></td>
			<td></td>
			<td>100</td>
		</tr>
		<tr>
			<td>Na-HEPES</td>
			<td></td>
			<td></td>
			<td>54.3</td>
		</tr>
		<tr>
			<td>Phenol Red</td>
			<td></td>
			<td></td>
			<td>1.1</td>
		</tr>
		<tr>
			<td>Pyruvic Acid</td>
			<td></td>
			<td></td>
			<td>3.6</td>
		</tr>
		<tr>
			<td>DL-Lactic Acid</td>
			<td></td>
			<td></td>
			<td>295</td>
		</tr>
	</tbody>
</table>

laser-assisted lentiviral gene delivery to mouse fertilized eggs

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

Development of isolated/transduced mouse fertilized eggs can be checked under the microscope daily (Figure 1). Healthy embryos develop into blastocyst within 3&#8211;4 days. In this protocol, 60&#8211;70% of untreated embryos develop into blastocyst23. Out of 114 laser-perforated transduced embryos, 54 developed into blastocyst (rate of 47%) and 46 blastocysts expressed GFP (46/54=85%)23.
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Laser-assisted Lentiviral Gene Delivery to Mouse Fertilized Eggs

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

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

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