The acoustic pore effect may not be applicable to the introduction of genes into tissues with lumen-forming or open structures. Acoustic pore effects on neural tube and limb ectoderm often cause diffuse gene expression due to diffusion of the injected microbubbles prior to sonication. Beyond these problems, the sound-well effect is an effective method for analyzing gene function in vivo and opens up a new field of gene transfer in medicine, molecular biology, and developmental biology. Author: T. Friedman et al, Translated by W. Qin et al. This experiment is from "Gene Transfer".
Operation method
Transfer of genes into embryos using the acoustic pore effect Move Transfer of genes into embryos using the acoustic pore effect Material reagents Ethanol (70 %) Expression carriers such as PEGFP (Clontech; 632319) with DNA concentrations of I.0 to 2.0ug/ul Dissolve the DNA in TE solution (pH 8.0). Chicken fertilized eggs Hank's solution (100 mmol/L HEPES, 140 mmol/L NaCl, 27 mmol/L KCl, 0.1% glucose, 0.34 m m o l / L Na2HPO4C, 0.5 m m o l / L CaCl2, 0.5 m m m o l / LMgCl2, pH ^ 7.4 ). 4 ) O P T I S O N : Microbubble reagent (A m e r s h a m H e a l t h 2707-03) Refrigerate at 2 to 8°C. Sodium chloride (N a C l ; 0. 9 % ) or leachate buffer (P B S ) T E solution (10 m m o l /L T ris--H C l , l m m o l /L E D T A , p H of 8.0) Instrumentation Black ink (Pelikan) Deterrent (w a t c h m a k e r's No. 5 straight valves) Glass microscope needles prepared from glass capillaries (C M ; Narishige) Pre-set the incubator at 38.5°C. Micropipettes Microscope Petri dish (35m m ) Scissors (straight, fine) Sonitron 1000N or Sonitron 2000N (R i c h M a e , Inola, Oklahoma or Nepagene in Japan) Injectors (5 ml) and 18 metering needles for the aspiration of fertilized eggs from egg white. 0.5 ml tube Vinyl Resin Tape Methods Preparation of DNA-microbubble mixtures 1. Take 5 ~ IOul of D N A storage solution (e.g. p E G F P I. 〇 ~ 2. Oug/pu) in a 0 -5m l tube. 2 . Add 10~15ul of 0 -9 % N a C l or P B S (D N A final concentration of 0.25~I.Oug/ul) to the tube. 3 . Shake the OPTISON gently until the solution is homogeneous. Add I0-20ul of microbubble solution to the tube containing DNA. Mix well and place on ice. The DNA-microbubble mixture can be used for 10 injections. Use within 2 h of preparation. Acoustic pore effect Example, used for gene transfer in HH20 ~2 1 stage chicken limb buds. 4 . Incubate chicken fertilized eggs at 38.5°C until HH 20 to 21 stage. 5 - Sterilize the redundant eggs with 70 % ethanol. Make a small hole in the rounded end of the egg. Aspirate 4 to 5 ml of egg white into a 5 ml syringe fitted with an 18-mm needle. 6 - Remove the eggshell with fine straight scissors, thus opening a window for later operations. 7 - Dilute the black ink with Hank's solution about 10 times. For observation of the chick embryo, 0 -05 to 0 -Im l diluted black ink is injected into the blastocyst layer with an Im l syringe fitted with a 26-metric needle. 8 . Remove the yolk membrane adjacent to the chick limb buds with fine watchmaker tweezers. If necessary, apply a few drops of Hank's solution. 9 . Mix the DNA-microbubble mixture evenly with a micropipette. Apply 2~3ul of DNA-microbubble mix to a 35 mm culture plate. Pipette 0.25~0.5ul1 of DNA-microbubble mix into a glass microneedle. Ensure that the microbubbles are in the DNA-microbubble mixture. 10- Inject the DNA-microbubble mixture into the limb bud mesenchyme (Figure 1C). Since the DNA-microbubble mixture will leak out from the injection site, the injection method can be adjusted according to the target organ. After injection, the presence of microbubbles at the injection site is ensured by observing white color (white color caused by reflected light) under a bright-field microscope. 11- Expose the injected limb bud site to ultrasound by gently touching the probe (3 mm diameter) of the ultrasonic instrument to the injection area. The parameters used were 2. OW/cm2 working period 2 0 %, and 60 s (Figs. 1A, B). After ultrasound exposure, the white color of the injection site disappeared. 12. After ultrasound exposure, the window was covered with vinyl tape and fertilized eggs were incubated. 13- Observe the site of sound pore effect DNA expression in chick embryos using LacZ (Fig. 2A , B) or green fluorescent protein (GFP) (Fig. 2C~E ). For more product details, please visit Aladdin Scientific website.
Injectors (Iml) and 26 metered-dose needles for the injection of ink.