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

Protocol

1. Perforation of Mouse Fertilized Eggs with XYClone Laser

1. Setup and calibrate XYClone laser according to the manufacturer's recommendation. Other lasers, typically used for in vitro fertilization, can be substituted for XYClone laser to perforate fertilized eggs.
   1. Attach the laser controller box wire to the laser apparatus on the microscope.
   2. 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.
   3. Looking through the eyepiece, perforate a test sample (e.g. dry-erase markings on a glass slide).
   4. 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.
2. 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.
3. Look through the microscope eyepiece and ensure that the embryo's zona pellucida is in focus and the laser LED light is visible.
4. Move the microscope stage to target the zona with the LED light/laser.
5. Using the computer software set XYClone laser to 250 μs.
6. Adjust the LED light size to desired dimensions (setting 5 in this experiment).
7. 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 μm (Figure 1).  
   NOTE: Aiming close to the polar body keeps laser away from the embryonic cell.
8. Allow fertilized eggs to recover for 2 h in the tissue culture incubator before moving to the next step.

2. Transduction of Mouse Fertilized Eggs Following XYClone Laser Perforation

1. Pipet 2 μL of concentrated lentivirus (greater than 1e8 TU/mL titer) into the 50 μL 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 μL is optimal for gene delivery.
2. Allow fertilized eggs to develop into blastocyst for 4 days in the incubator. No need to change the media.

3. Non-surgical Transfer of Transduced Mouse Embryos to Pseudo-pregnant Mice

1. Use pseudopregnant mice, 3.5 day after mating.
2. Use Non-Surgical Embryo Transfer (NSET) device to implant mouse embryos into pseudopregnant mice.
   1. Using a surgical or dissecting microscope, insert a vaginal speculum to visualize the cervix.
   2. Insert the Non-Surgical Embryo Transfer (NSET) device approximately 5 mm into the cervix and deposit embryos in a volume of approximately 2 μL.
      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.
3. Transfer 10–15 healthy blastocyst in 2 μL volume of KSOM to each pseudopregnant mice.
4. Continue to monitor and measure the weight gain in pseudopregnant mice in following days to determine whether the NSET was successful.
5. Recover pups by allowing the pregnant mice to give birth naturally or performing a caesarean section 17 days after the embryo transfer. A C-section is often necessary if very few embryos are present and they grow too large for natural birth.
6. Collect tissue from pups for genotyping to determine the rate of transgenesis.

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Results

Figure 1: Laser-Treatment of Mouse Fertilized Eggs. Examples of perforating the zona to produce a hole vs thinning of the zona.

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Materials

Name	Company	Catalog Number	Comments
CD510B-1 plasmid	System Biosciences	CD510B-1	used to package the lentivirus expressing EF1a-copGFP
Dimethylpolysiloxane	Sigma	DMPS5X	culturing embryos
XYClone Laser	Hamilton Thorne Biosciences		perforating mouse fertilized eggs
Non-Surgical Embryo Transfer (NSET) Device	ParaTechs	60010	NSET of embryos
KSOM medium	Millipore	MR-020P-5F	culturing embryos
Composition of KSOM:			mg/100mL
NaCl			555
KCl			18.5
KH2PO4			4.75
MgSO4 7H2O			4.95
CaCl2 2H2O			25
NaHCO3			210
Glucose			3.6
Na-Pyruvate			2.2
DL-Lactic Acid, sodium salt			0.174mL
10 mM EDTA			100µL
Streptomycin			5
Penicillin			6.3
0.5% phenol red			0.1mL
L-Glutamine			14.6
MEM Essential Amino Acids			1mL
MEM Non-essential AA			0.5mL
BSA			100