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 for transduction. Standard protocols2 were used to produce and titer lentiviruses at titers higher than 1e8 transducing units per mL (TU/mL). 
	NOTE: Use caution and bleach all material that have been in contact with lentiviruses. Refer to your institute&#39;s guidelines for safe use and handling of lentiviruses.</li>
	<li>Setup breeding pairs of desired mouse strain the day before harvesting embryos. In this experiment, C57BL/6J strain of mice was used. Female mice used for ovaries and oviduct collection were not treated with any hormones for superovulation.</li>
	<li>2&#8211;24 h prior to harvesting mouse fertilized eggs, prepare several Potassium Simplex Optimized Medium24 (KSOM) drop plates as follows: add a 50 &#956;L drop of KSOM medium to the middle of a 35 mm tissue culture-treated dish and cover with 2 mL of Dimethylpolysiloxane (DMPS5X) and place at 37 oC, 5% CO2, 5% O2 and 90% N2 to equilibrate. 
	NOTE: Use disposable sterile plates and discard following exposure to lentiviruses.</li>
	<li>Prepare 1x solution of hyaluronidase in M2 medium from stock solution (100x, 30 mg/mL, store at -20 &#176;C). 100x stock solution was prepared in water.</li>
	<li>24 h post laser-assisted transduction of mouse fertilized eggs, set up breeding pairs between vasectomized male and female mice to prepare pseudopregnant female mice.</li>
</ol>
2. Isolation of Mouse Fertilized Eggs
<ol>
	<li>16&#8211;24 h post mating, select female mice with vaginal plugs indicative of successful mating and humanely euthanize25. The mice used in this study were euthanized by cervical dislocation under deep CO2 or isoflurane inhalation.</li>
	<li>Isolate ovaries and oviducts according to standard protocols25.</li>
	<li>Transfer the ovaries and oviducts to a 35 mm dish containing M2 media.</li>
	<li>For each ovary, tear ampulla apart to release fertilized eggs surrounded by cumulus cells.</li>
	<li>Transfer fertilized eggs and cumulus cells to a 35 mm dish containing 1x hyaluronidase in M2 medium and incubate at room temperature for 3&#8211;5 min.</li>
	<li>Pipet fertilized eggs up and down to release the cumulus cells.</li>
	<li>Transfer fertilized eggs to a 35 mm dish containing M2 media and pipet up and down to wash off hyaluronidase.</li>
	<li>Transfer fertilized eggs to a 35 mm dish containing KSOM and pipet up and down to wash off the remaining M2 media and hyaluronidase.</li>
	<li>Transfer fertilized eggs to the prepared KSOM drop plates from step 1.3. 
	NOTE: Mouse fertilized eggs in KSOM must be kept in a tissue culture incubator at 37 &#176;C, 5% CO2, 5% O2 and 90% N2 to equilibrate. Removing plates from the incubator for longer than 15 min will result in damage to embryos.</li>
	<li>Allow fertilized eggs to recover for 2 h in the incubator before moving to the next step.</li>
</ol>
3. Perforation of Mouse Fertilized Eggs with XYClone Laser
<ol>
	<li>Setup and calibrate XYClone laser according to the manufacturer&#39;s recommendation. Other lasers, typically used for in vitro fertilization, can be substituted for XYClone laser to perforate fertilized eggs.
	<ol>
		<li>Attach the laser controller box wire to the laser apparatus on the microscope.</li>
		<li>Attach the laser controller box to the computer running the laser software via a USB port. 3.1.3. Plug in the laser controller and switch it on.</li>
		<li>Looking through the eyepiece, perforate a test sample (e.g. dry-erase markings on a glass slide).</li>
		<li>Use a small screw driver (included in the laser kit) to adjust the X and Y position of the laser to match the LED light visible through the microscope eyepiece and calibrate the laser.</li>
	</ol>
	</li>
	<li>Place a KSOM drop plate containing mouse fertilized eggs on the microscope stage. Do not keep the plate outside of the incubator for longer than 15 min.</li>
	<li>Look through the microscope eyepiece and ensure that the embryo&#39;s zona pellucida is in focus and the laser LED light is visible.</li>
	<li>Move the microscope stage to target the zona with the LED light/laser.</li>
	<li>Using the computer software set XYClone laser to 250 &#956;s.</li>
	<li>Adjust the LED light size to desired dimensions (setting 5 in this experiment).</li>
	<li>Perforate the zona of each fertilized egg thrice with the laser. The zona can be either pierced or thinned. Using the above settings, the laser will produce a hole with a diameter of 10 &#956;m (Figure 2). 
	NOTE: Aiming close to the polar body keeps laser away from the embryonic cell.</li>
	<li>Allow fertilized eggs to recover for 2 h in the tissue culture incubator before moving to the next step.</li>
</ol>
4. Transduction of Mouse Fertilized Eggs Following XYClone Laser Perforation
<ol>
	<li>Pipet 2 &#956;L of concentrated lentivirus (greater than 1e8 TU/mL titer) into the 50 &#956;L KSOM drop. Do not pipet up and down. Fertilized eggs readily attach to the pipet tip. Based on our experience, 1e5-5e5 transducing units of lentivirus in a volume less than 3 &#956;L is optimal for gene delivery.</li>
	<li>Allow fertilized eggs to develop into blastocyst for 4 days in the incubator. No need to change the media.</li>
</ol>
5. Non-surgical Transfer of Transduced Mouse Embryos to Pseudo-pregnant Mice
<ol>
	<li>Use pseudopregnant mice, 3.5 day after mating, prepared in step 1.5.</li>
	<li>Use Non-Surgical Embryo Transfer (NSET) device to implant mouse embryos into pseudopregnant mice.
	<ol>
		<li>Using a surgical or dissecting microscope, insert a vaginal speculum to visualize the cervix.</li>
		<li>Insert the Non-Surgical Embryo Transfer (NSET) device approximately 5 mm into the cervix and deposit embryos in a volume of approximately 2 &#956;L. 
		NOTE: A sterile NSET device is used for each transfer and discarded after the procedure. All reagents used in the manipulation of the embryos must be sterile.</li>
	</ol>
	</li>
	<li>Transfer 10&#8211;15 healthy blastocyst in 2 &#956;L volume of KSOM to each pseudopregnant mice.</li>
	<li>Continue to monitor and measure the weight gain in pseudopregnant mice in following days to determine whether the NSET was successful.</li>
	<li>Recover pups by allowing the pregnant mice to give birth naturally or performing a caesarean section 17 days after the embryo transfer26. A C-section is often necessary if very few embryos are present and they grow too large for natural birth.</li>
	<li>Collect tissue from pups for genotyping to determine the rate of transgenesis27.</li>
</ol>

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<li>Horii, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Validation+of+microinjection+methods+for+generating+knockout+mice+by+CRISPR%2FCas-mediated+genome+engineering%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">Validation of microinjection methods for generating knockout mice by CRISPR/Cas-mediated genome engineering.</a> Scientific Reports. 4, 4513(2014).</li>
<li>Hamra, F. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Production+of+transgenic+rats+by+lentiviral+transduction+of+male+germ-line+stem+cells%5BTitle%5D)+AND+%22Proceedings+of+National+Academy+of+Science+U+S+A%22%5BJournal%5D)">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/pubmed?term=((Production+of+transgenic+rats+via+lentiviral+transduction+and+xenogeneic+transplantation+of+spermatogonial+stem+cells%5BTitle%5D)+AND+%22Biology+of+Reproduction%22%5BJournal%5D)">Production of transgenic rats via lentiviral transduction and xenogeneic transplantation of spermatogonial stem cells.</a> Biology of Reproduction. 79 (6), 1121-1128 (2008).</li>
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</ol>

The authors have nothing to disclose.

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 stages of development and brand lentiviruses as powerful tools for gene delivery.
Laser-assisted lentiviral transgenesis is an effective and easy-to-use method for gene expression in vivo. This method can be used for in vivo protein production, expression of genetically encoded indicators, or functional studies. Compared to other methods, laser-assisted lentiviral gene delivery is as effective as pronuclear microinjection but requires no technical skills or costly microinjection workstations. The XYClone laser is small, portable, and can easily be shared among several laboratories.
Laser-assisted lentiviral gene delivery is stable and not transient, as in electroporation or photoporation of fertilized eggs. Transient gene delivery is more advantages for delivery of CRISPR-Cas9 components or recombinases since extended expression could lead to aberrant consequences. In this protocol, the integrase deficient lentiviruses (IDLVs) can be substituted for transient transduction of mouse fertilized eggs. IDLVs retain lentiviral infectivity but only retain a fraction (less than 8%) of integrative capability of lentiviruses29. Laser-assisted lentiviral transgenesis is an additional gene delivery option for users to evaluate based on their desired outcome.
The major disadvantage of lentiviral transduction is the random insertion of the delivered gene. Also, cells within the same embryo could host multiple lentiviral integrations that leads to mosaicism in the transgenic. Genotyping of the progeny and multiple rounds of planned breeding is necessary to establish a single locus transgenic animal. Similar breeding strategies are also employed in conventional transgenesis methods30.
A critical step in this protocol is the length of time spent on laser perforation. Mouse fertilized eggs cultured in KSOM cannot be kept outside of the incubator for longer than 15 min. A novice user should move the fertilized eggs to M2 medium, use Advanced KSOM Embryo Medium, or limit the number of embryos per plate. According to our result, 47% of transduced cultured mouse fertilized eggs develop into blastocysts23. Therefore, culturing 30-40 fertilized eggs per plate will ensure adequate number of transduced blastocysts in one plate for transfer into pseudopregnant mice.
Laser-assisted perforation of the mouse fertilized egg zona may also be applicable to the zona of other species and allow entry for other types of viruses or transfection reagents.

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>&lt;strong&gt;KSOM medium&lt;/strong&gt;</td><td>Millipore</td><td>MR-020P-5F</td><td>culturing embryos</td></tr><tr><td>&lt;strong&gt;Composition of KSOM:&lt;/strong&gt;</td><td></td><td></td><td>&lt;strong&gt;mg/100mL&lt;/strong&gt;</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>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td></td><td></td><td>4.75</td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt; 7H&lt;sub&gt;2&lt;/sub&gt;O</td><td></td><td></td><td>4.95</td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt; 2H&lt;sub&gt;2&lt;/sub&gt;O</td><td></td><td></td><td>25</td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</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>10 mM EDTA</td><td></td><td></td><td>100&amp;micro;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>&lt;strong&gt;M2 medium&lt;/strong&gt;</td><td>Millipore</td><td>MR-015-D</td><td>culturing embryos</td></tr><tr><td>&lt;strong&gt;Composition of M2:&lt;/strong&gt;</td><td></td><td></td><td>&lt;strong&gt;mg/100mL&lt;/strong&gt;</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.
The mouse embryos were laser-perforated on the day of harvest. The optimal setting for the laser treatment was 3 holes per fertilized egg and 250 ms. The laser should be aimed to thin the zona instead of creating a hole (Figure 2). This allows the developing embryos to benefit from an intact encapsulating zona pellucida while becoming permissive to lentiviral transduction. In these experiments, mouse embryos were transduced with a recombinant lentivirus that expressed copepod GFP (abbreviated GFP) from an elongation factor 1a promoter. For transduction, 2 uL of lentivirus was introduced into the culture media. Increasing the amount of virus, directly affected the number of transduced embryos that expressed GFP while adversely affecting embryo development into blastocyst23. Viral volumes larger than 10% of KSOM drop volume would also adversely affect blastocyst formation (e.g. greater than 5 &#956;L of virus in a 50 &#956;L KSOM drop).
To validate the viability, transduced mouse embryos were non-surgically transferred to pseudopregnant mice. Six separate NSET events, transferring a total of 58 blastocyst, resulted in 9 GFP transgenic pups out of total of 12, yielding 75% rate of transgenesis 23. The NSET protocol is reported to yield 30-35% live births28 compared to 21% yield that were observed in our experiments (12/58). The lentiviral treatment may have played a role in embryo development and contributed to the low embryo transfer rate. Pups with multiple lentiviral integrations and high expression of GFP were visually green under a blue LED lamp (465-470 nm) (Figure 3). The number of incorporated GFP copies ranged from 0-6 copies and was determined by performing qualitative PCR on isolated chromosomal DNA from pup tissue23.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58327/58327fig1.jpg" /> 
Figure 1: Development of Transduced Mouse Fertilized Eggs in Culture. The C57BL/6J mouse fertilized eggs were monitored on Day 0 (harvested fertilized egg), Day 1 (two-cell), Day 2 (8-16 cells), Day 3 (morula), and Day 4 (blastocyst). Embryos were transduced with a lentivirus carrying a GFP gene. Presence of Fluorescence in transduced embryos became evident by Day 2-3. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/58327/58327fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58327/58327fig2.jpg" /> 
Figure 2: Laser-Treatment of Mouse Fertilized Eggs. Examples of perforating the zona to produce a hole vs thinning of the zona.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58327/58327fig3.jpg" /> 
Figure 3: Transgenic Mice Expressing GFP. Dark Reader (DR Spot Lamp, DRSL-9S) was used to visualize GFP positive pups in a dark environment. Non-transgenic control pups were added to the group for contrast. Resulting pups (red arrows) were genotyped and contained from 0-6 copies of the lentiviral gene/GFP.

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Laser-assisted Lentiviral Gene Delivery to Mouse Fertilized Eggs | Biology | Video

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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7.6K Views.  National Institute of Environmental Health Sciences. 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.

Video: Laser-assisted Lentiviral Gene Delivery to Mouse Fertilized Eggs

Laser-assisted Cytoplasmic Microinjection in Livestock Zygotes

Cytoplasmic microinjection into one-cell embryos is a very powerful technique. As an example, it enables the delivery of genome editing tools that can create genetic modifications that will be present in every cell of an adult organism. It can also be used to deliver siRNA, mRNAs or blocking antibodies to study gene function in preimplantation embryos. The conventional technique for microinjecting embryos used in rodents consists of a very thin micropipette that directly penetrates the plasma membrane when advanced into the embryo. When this technique is applied to livestock animals it usually results in low efficiency. This is mainly because in contrast to mice and rats, bovine, ovine, and porcine zygotes have a very dark cytoplasm and a highly elastic plasma membrane that makes visualization during injection and penetration of the plasma membrane hard to achieve. In this protocol, we describe a suitable microinjection method for the delivery of solutions into the cytoplasm of cattle zygotes that has proved to be successful for sheep and pig embryos as well. First, a laser is used to create a hole in the zona pellucida. Then a blunt-end glass micropipette is introduced through the hole and advanced until the tip of the needle reaches about 3/4 into the embryo. Then, the plasma membrane is broken by aspiration of cytoplasmic content inside the needle. Finally, the aspirated cytoplasmic content followed by the solution of interest is injected back into the embryonic cytoplasm. This protocol has been successfully used for the delivery of different solutions into bovine and ovine zygotes with 100% efficiency, minimal lysis, and normal blastocysts development rates.

This protocol shows how to perform cytoplasmic microinjection in farm animal zygotes. This technique can be used to deliver any solution into the one-cell embryo such as genome editing tools to generate knockout animals.

Cytoplasmic microinjection into one-cell embryos is a very powerful technique. As an example, it enables the delivery of genome editing tools that can ...

Windowing Chicken Eggs for Developmental Studies

The study of development has been greatly aided by the use of the chick embryo as an experimental model.  The ease of accessibility of the embryo has allowed for experiments to map cell fates using several approaches, including chick quail chimeras and focal dye labeling.  In addition, it allows for molecular perturbations of several types, including placement of protein-coated beads and introduction of plasmid DNA using in ovo electroporation.  These experiments have yielded important data on the development of the central and peripheral nervous systems.  For many of these studies, it is necessary to open the eggshell and reclose it without perturbing the embryo's growth.  The embryo can be examined at successive developmental stages by re-opening the eggshell.  While there are several excellent methods for opening chicken eggs, in this article we demonstrate one method that has been optimized for long survival times.  In this method, the egg rests on its side and a small window is cut in the shell.  After the experimental procedure, the shell is used to cover the egg for the duration of its development.  Clear plastic tape overlying the eggshell protects the embryo and helps retain hydration during the remainder of the incubation period.  This method has been used beginning at two days of incubation and has allowed survival through mature embryonic ages.

The ease of accessibility of the embryo has allowed for experiments to map cell fates using several approaches, including chick quail chimeras and focal dye labeling. In this article we demonstrate one egg preparation method that has been optimized for long survival times.

The study of development has been greatly aided by the use of the chick embryo as an experimental model.  The ease of accessibility of the embryo has ...

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

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

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

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

Obtaining Eggs from Xenopus laevis Females

The eggs of Xenopus laevis intact, lysed, and/or fractionated are useful for a wide variety of experiments. This protocol shows how to induce egg laying, collect and dejelly the eggs, and sort the eggs to remove any damaged eggs.

The eggs of Xenopus laevis intact, lysed, and/or fractionated are useful for a wide variety of experiments. This protocol shows how to induce egg laying, collect and dejelly the eggs, and sort the eggs to remove any damaged eggs.

The eggs of Xenopus laevis intact, lysed, and/or fractionated are useful for a wide variety of experiments. This protocol shows how to induce egg laying, ...

Subretinal Injection of Gene Therapy Vectors and Stem Cells in the Perinatal Mouse Eye

The loss of sight affects approximately 3.4 million people in the United States and is expected to increase in the upcoming years.1 Recently, gene therapy and stem cell transplantations have become key therapeutic tools for treating blindness resulting from retinal degenerative diseases. Several forms of autologous transplantation for age-related macular degeneration (AMD), such as iris pigment epithelial cell transplantation, have generated encouraging results, and human clinical trials have begun for other forms of gene and stem cell therapies.2 These include RPE65 gene replacement therapy in patients with Leber's congenital amaurosis and an RPE cell transplantation using human embryonic stem (ES) cells in Stargardt's disease.3-4 Now that there are gene therapy vectors and stem cells available for treating patients with retinal diseases, it is important to verify these potential therapies in animal models before applying them in human studies. The mouse has become an important scientific model for testing the therapeutic efficacy of gene therapy vectors and stem cell transplantation in the eye.5-8 In this video article, we present a technique to inject gene therapy vectors or stem cells into the subretinal space of the mouse eye while minimizing damage to the surrounding tissue.

This surgical technique illustrates the injection of gene therapy vectors and stem cells into the subretinal space of the mouse eye.

The loss of sight affects approximately 3.4 million people in the United States and is expected to increase in the upcoming years.1 Recently, gene therapy ...

Medicine

CRISPR-mediated Loss of Function Analysis in Cerebellar Granule Cells Using In Utero Electroporation-based Gene Transfer

Brain malformation is often caused by genetic mutations. Deciphering the mutations in patient-derived tissues has identified potential causative factors of the diseases. To validate the contribution of a dysfunction of the mutated genes to disease development, the generation of animal models carrying the mutations is one obvious approach. While germline genetically engineered mouse models (GEMMs) are popular biological tools and exhibit reproducible results, it is restricted by time and costs. Meanwhile, non-germline GEMMs often enable exploring gene function in a more feasible manner. Since some brain diseases (e.g., brain tumors) appear to result from somatic but not germline mutations, non-germline chimeric mouse models, in which normal and abnormal cells coexist, could be helpful for disease-relevant analysis. In this study, we report a method for the induction of CRISPR-mediated somatic mutations in the cerebellum. Specifically, we utilized conditional knock-in mice, in which Cas9 and GFP are chronically activated by the CAG (CMV enhancer/chicken &#223;-actin) promoter after Cre-mediated recombination of the genome. The self-designed single-guide RNAs (sgRNAs) and the Cre recombinase sequence, both encoded in a single plasmid construct, were delivered into cerebellar stem/progenitor cells at an embryonic stage using in utero electroporation. Consequently, transfected cells and their daughter cells were labeled with green fluorescent protein (GFP), thus facilitating further phenotypic analyses. Hence, this method is not only showing electroporation-based gene delivery into embryonic cerebellar cells but also proposing a novel quantitative approach to assess CRISPR-mediated loss-of-function phenotypes.

CRISPR-mediated Loss of Function Analysis in Cerebellar Granule Cells Using In Utero Electroporation-based Gene Transfer

Conventional loss-of-function studies of genes using knockout animals have often been costly and time-consuming. Electroporation-based CRISPR-mediated somatic mutagenesis is a powerful tool to understand gene functions in vivo. Here, we report a method to analyze knockout phenotypes in proliferating cells of the cerebellum.

Brain malformation is often caused by genetic mutations. Deciphering the mutations in patient-derived tissues has identified potential causative factors ...

Developmental Biology

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

Genetic modification of specific regions of the developing mammalian brain is a very powerful experimental approach. However, generating novel mouse mutants is often frustratingly slow. It has been shown that access to the mouse brain developing in utero with reasonable post-operatory survival is possible. Still, results with this procedure have been reported almost exclusively for the most superficial and easily accessible part of the developing brain, i.e. the cortex. The thalamus, a narrower and more medial region, has proven more difficult to target. Transfection into deeper nuclei, especially those of the hypothalamus, is perhaps the most challenging and therefore very few results have been reported. Here we demonstrate a procedure to target the entire hypothalamic neuroepithelium or part of it (hypothalamic regions) for transfection through electroporation. The keys to our approach are longer narcosis times, injection in the third ventricle, and appropriate kind and positioning of the electrodes. Additionally, we show results of targeting and subsequent histological analysis of the most recessed hypothalamic nucleus, the mammillary body.

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

Despite the functional and medical importance of the hypothalamus, in utero genetic manipulation of its development has rarely been attempted. We show a detailed procedure for in utero electroporation into the mouse hypothalamus and show representative results of total and partial (regional) hypothalamic transfection.

Genetic  modification of specific regions of the developing mammalian brain is a very  powerful experimental approach. However, generating novel mouse ...

Neuroscience

CRISPR/Cas9-Mediated Highly Efficient Gene Targeting in Embryonic Stem Cells for Developing Gene-Manipulated Mouse Models

The CRISPR/Cas9 system has made it possible to develop genetically modified mice by direct genome editing using fertilized zygotes. However, although the efficiency in developing gene-knockout mice by inducing small indel mutation would be sufficient enough, the efficiency of embryo genome editing for making large-size DNA knock-in (KI) is still low. Therefore, in contrast to the direct KI method in embryos, gene targeting using embryonic stem cells (ESCs) followed by embryo injection to develop chimera mice still has several advantages (e.g., high throughput targeting in vitro, multi-allele manipulation, and Cre and flox gene manipulation can be carried out in a short period). In addition, strains with difficult-to-handle embryos in vitro, such as BALB/c, can also be used for ESC targeting. This protocol describes the optimized method for large-size DNA (several kb) KI in ESCs by applying CRISPR/Cas9-mediated genome editing followed by chimera mice production to develop gene-manipulated mouse models.

Here we present a protocol for developing genetically modified mouse models using embryonic stem cells, especially for large DNA knock-in (KI). This protocol is tuned up using CRISPR/Cas9 genome editing, resulting in significantly improved KI efficiency compared with the conventional homologous recombination-mediated linearized DNA targeting method.

The CRISPR/Cas9 system has made it possible to develop genetically modified mice by direct genome editing using fertilized zygotes. However, although the ...

Lentiviral Vector-Based Perivitelline Injection: A Method to Transduce the Gene of Interest Into Fertilized Single-Cell Mouse Oocytes

Source: Dussaud, S. et al., <a href="https://www-jove-com.bdigitaluss.remotexs.co/v/57609">Lentiviral Mediated Production of Transgenic Mice: A Simple and Highly Efficient Method for Direct Study of Founders.</a> J. Vis. Exp. (2018).
This video describes the pronuclear injection of lentiviral vectors into the perivitelline space of a fertilized mouse embryo. The lentivirus contains the transgene, which is incorporated into the genome of the fertilized embryo, eventually giving rise to transgenic mice.

Source: Dussaud, S. et al., Lentiviral Mediated Production of Transgenic Mice: A Simple and Highly Efficient Method for Direct Study of Founders. J. Vis. ...

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Biological Techniques

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Transduction

Laser-Assisted Lentiviral Gene Delivery: A Technique to Permeabilize Mouse Fertilized Eggs to Facilitate Lentiviral Gene Delivery

Source: Martin, N. P., et al. <a href="https://www-jove-com.bdigitaluss.remotexs.co/v/58327">Laser-assisted Lentiviral Gene Delivery to Mouse Fertilized Eggs.</a> J. Vis. Exp. (2018).
In this video, we demonstrate the laser-assisted permeation of the protective layer of zona pellucida in mouse fertilized eggs for facilitating lentiviral gene delivery. Lentivirus enables the generation of transgenic animals with a gene of interest stably integrated into their genome.

Source: Martin, N. P., et al. Laser-assisted Lentiviral Gene Delivery to Mouse Fertilized Eggs. J. Vis. Exp. (2018).
In this video, we demonstrate the ...

Laser-assisted Lentiviral Gene Delivery to Mouse Fertilized Eggs

Summary

Explore More Videos

Laser-assisted Cytoplasmic Microinjection in Livestock Zygotes

Windowing Chicken Eggs for Developmental Studies

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

Obtaining Eggs from Xenopus laevis Females

Subretinal Injection of Gene Therapy Vectors and Stem Cells in the Perinatal Mouse Eye

CRISPR-mediated Loss of Function Analysis in Cerebellar Granule Cells Using In Utero Electroporation-based Gene Transfer

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

CRISPR/Cas9-Mediated Highly Efficient Gene Targeting in Embryonic Stem Cells for Developing Gene-Manipulated Mouse Models

Lentiviral Vector-Based Perivitelline Injection: A Method to Transduce the Gene of Interest Into Fertilized Single-Cell Mouse Oocytes

Laser-Assisted Lentiviral Gene Delivery: A Technique to Permeabilize Mouse Fertilized Eggs to Facilitate Lentiviral Gene Delivery