This chapter describes the use of lentiviral vectors to transfer genes and small interfering RNA (siR N A ) expression cassettes to preimplantation crystalline embryos. This technique has enabled the rapid development of rapidly transgenic animals and transgenic down-regulated animals
Author: T. Friedman et al., Translated by W. Qin et al. This experiment is from "Gene Transfer".
Operation method
Oviductal hyaline removal and sub-band injections by Move Oviductal hyaline removal and sub-band injections by Materials reagents Acidic Tyrode solution Mix the following in 80 ml of water: 0.8 g NaCi, 0.02 g KCl, 0.024 g CaCl2,2 H20, 0 - Olg MgCl2.6 H2O, 0 - Ig glucose and 0.4 g polyvinylpyrrolidone (PVP). The pH was adjusted to 2.5 in a final volume of 100 ml. Gonadotropins derived from pregnant mare serum (PMS; 25IU/m l saline solution XSigma-Aldrich G 4527) Human chorionic gonadotropin (hCG; 25IU/m l salt solution) (Sigma-Aldrich G 8554) Hyaluronidase M2 solution (Specialty Media MR-051-F) Medium M2 (Special Medium MR-015-D) M16 (Special Medium MR-010-D) Mouse B6D2F1 Female (6-8 weeks old; Haarlan) Pseudopregnant female, synchronized (2.5d post mating [dpc]; 0.5dpc selected for subcutaneous injection) Mineral oil (Fischer 0121-1) Virus for genetic modification ( 109 viral particles/ml) Instrumentation Round coverslips for cell culture Prepare the pipette by gently pulling the borosilicate glass capillary over the flame of a Bunsen burner until the outer layer of the pipette is pulled out to a diameter of 80 to 120 um. Use a 10ul capillary micropipette for this step (VWR 53432 to 728). Prepare the borosilicate glass capillary by gently pulling it (gently over a Bunsen burner flame) until the inner layer of the tip is about 200um in diameter. use this 50ul micropipette for this step (VWR 53432 ~ 783). Incubate, preset to 37°C , 5 % C0 2 Inverted microscope Microcentrifuge Micromanipulation equipment Cell-trim oil pump (Eppendorf) used to regulate needle holding Manual Micromanipulator Pair (Lightz Acsol) for needle holding and injections Micro pipette (ICSI, sterile) (M I C -cust-0, I D 4~5um, O D 5~6um , bevellength 50°C [8~9fim] with a spike H u m a g e n , Virginia) Screw-actuated syringe (S A S 11/2- E [Z M S ] ) used to operate injection needles (30 G ) Pasteur pipette binoculars Surgical instruments: blunt curved forceps (2), fine point forceps (2), small scissors Methods Preparation of mice for crystal embryo harvesting 1. Inject 8~I2 B6D2F l female rats (6~8 weeks old, Harrlan) with 5 units of PMS intraperitoneally. 2- Just 48 h injection of 5 units of hCG. Transfer females to male cages (one female and one male). 3. Perform oval zona pellucida removal or subcutaneous injection. Removal of the zona pellucida a. 48 h after hCG injection, surgically collect the oviducts under aseptic conditions and transfer to a drop of 50 ul of mineral oil-covered M 2. Insert a funnel tube containing a pure 30 G needle and blow out the embryos (2 to 4 cell periods) with M 2 medium. Wash the embryos with several successive droplets of M 2 by pipetting up and down 3 to 4 times. Repeat this step with M 16 medium. Working quickly, incubate the embryos at 37°C until the next step. b. To remove the egg zona pellucida, transfer the crystal embryo to a drop of acidic T y r o d e solution, pipette up and down several times, and then transfer to a second drop of acidic T y r o d e solution. Incubate at room temperature until the egg zona pellucida is dissolved (30 s ~ I m i n ). The crystal embryos were washed twice with a Pasteur pipette in a mineral oil-covered 50ul M2 drop. Repeat this rinsing procedure 3 times in a mineral oil-covered droplet of M 16 medium. c. For transduction of viral particles, use a virus titer of IO9 viral particles/m i. Dilute the virus 100-fold in M 16. Transfer the crystals into a 30/xl droplet of diluted virus solution by pipetting up and down several times and then transferring individual crystals into a separate IOul of mineral oil-covered diluted virus droplet. Incubate for 48 h at 37°C, 5 % C02. d. After 48 h, the crystalline embryos should develop into blastomeres. A transfer needle was used to transfer the developed blastocysts to the uterus of 2.5 dpc sham pregnant females (Hogan etal. 1994). Subcutaneous injection a- 24 h after h C G injection, the fallopian tubes were surgically collected under aseptic conditions and transferred to a mineral oil-covered 50 ul droplet of M 2 liquid. A pair of fine forceps is used to gently release the crystalline embryo (unicellular stage) from the swollen juxtapetalum (upper part of the fallopian tube), and then the crystalline embryo is transferred to hyaluronidase M2 solution, where the enzyme is used to digest the cells that have accumulated next to the crystalline embryo. The embryo is transferred to fresh M2 medium to wash off the hyaluronidase solution and then placed in a drop of 50 ul mineral oil-covered M16 medium. b - Thaw a viral pre-fraction ( 109 viral particles/m l ) by brief centrifugation (5 s at high speed) to prevent cell debris from clogging the tip of the micropipette. c. Prepare for microprocessing. After mounting the syringe needle, dispense 5ul of virus suspension into a cell culture with a round coverslip, using negative pressure through the needle. If the needle becomes clogged, release debris with a brief positive airflow. Transfer single-cell crystals to a drop of M 16 medium covered with mineral oil. Microinjection can be performed with homemade micropipettes (Loiset al.2002) or customized sterile ICSI micropipettes. d- Injections are performed under an inverted microscope. Attach the micropipette containing the virus to the micromanipulator and lower the tip into the mineral oil until the tip is visible in the field of view. Turn on the positive airflow while holding the needle to the crystalline embryo and gently push the micropipette tip through the band layer to the periplasmic space without damaging the crystalline embryo cell membrane. Keep the tip in the band layer to the periplasmic space for 5 to 10 s. Some increase in space indicates the presence of positive pressure. Incubate crystal embryos in mineral oil-covered M 16 medium at 37°C with 5 % CO2 until transplantation. e. Immediately transfer crystalline embryos into the oviducts of 0.5d pc synchronized pseudopregnant females or incubate crystalline embryos for 2 to 3d until follicle formation, then transfer into the uterus of 2.5d pc synchronized pseudopregnant females. For more product details, please visit Aladdin Scientific website.