1. Preparation of Protamine Sulfate (PS) Coated Dishes
2. Obtain Sea Urchin Gametes and Immobilize the Eggs on a PS-coated Dish for Microinjection
3. Row the Eggs
4. Microinjection of the Sea Urchin Zygotes
GFP and mCherry reporter constructs were in vitro transcribed and microinjected into the newly fertilized eggs. Embryos were incubated at 15 °C for 24 hr (until the blastula stage) and imaged using Zeiss Observer Z1 microscope. Injection of reporter constructs did not lead to any developmental defects (Figure 6). For quantification of fluorescent signals, image acquisition was performed at low magnification (100X) to maximally capture fluorescent pixels (Figures 6D-F). Fluorescent signals were quantified using Axiovision 4.8.2.0. The standard errors for the intensity of fluorescent signals within the population of 100-200 blastulae did not exceed 1.5% (data not shown).
Figure 1. The microinjection setup. (A) A PS-coated dish with immobilized eggs is located on the stage of (B) an inverted microscope. Microinjection needle is attached to (C) a needle holder which is connected to a (D) pressure unit. Movement in the x-, y-, and z-dimensions is directed with using (E) the micromanipulator or (F) the coarse manipulator. (G) A screw driver wrapped with paper towels is used to gently tap the microscope stage to induce slight trembling to facilitate needle entry into the newly fertilized egg.
Figure 2. Spawning of the sea urchins. (A) Sea urchin is induced to shed by intracoelomic injection of 0.5% KCl. The needle points to the peristomial membrane surrounding the mouth of the animal, the only soft part of the animal. (B) Female sea urchin is placed with its gonapores immersed in the sea water in a plastic beaker to collect yellow eggs. (C) White sperm is released from gonapores of the male sea urchin.
Figure 3. Mouth pipette. A mouth pipette consists of three parts: (A) Glass micropipette needle, (B) plastic tubing, and (C) a sterile filtered P20 or P200 tip. The glass micropipette is inserted into the plastic tubing which is connected to the P20 or P200 mouth piece.
Figure 4. Microinjection needle. (A) The microinjection needle is pulled using a needle puller with a specific setting. The setting of the needle puller needs to be empirically determined. Using a Narishege PC-10 needle puller, we use 72.8 °C for heat setting 1 and 83.7 °C for heat setting 2. Using the scratch on the plate we break the tip of the needle to facilitate solution flow. (B) The good needle should have a length of 500-600 μm from a width of 50 μm at the shoulder to 5 μm at the tip.
Figure 5. Injection of the newly fertilized sea urchin eggs. Dejellied eggs were rowed on a PS-coated dish and fertilized. Newly fertilized eggs were injected one by one while moving the microscope stage along the line of zygotes (from top to bottom). (A) Injection bolus is clearly seen to be forming inside the newly fertilized egg at the tip of the needle. (B) Transparent fertilization envelope is formed upon fertilization. (C) Sperm appear as small black dots in the background. Scale bar is 50 μm. Click here to view larger image.
Figure 6. Microinjected zygotes developed into blastulae without developmental or morphological defects. Newly fertilized eggs were injected with fluorescent markers which allow them to be clearly distinguished from the uninjected embryos (shown by arrows). (A-C) Embryos imaged at 400X magnification. (D-F) Embryos are imaged at 100X magnification to capture most of the emitted fluorescence signals for reliable quantification. After 24 hr post fertilization, blastulae were collected and treated with 2x sea water for 2 min to immobilize the swimming embryos3, followed by 1x sea water wash. Images were acquired with Zeiss Observer Z1 microscope and AxioCam monochromic camera. (A,D) DIC images of the blastulae. (B,E) Overlay images of embryos injected with in vitro transcribed mCherry in fluorescence and DIC channels. (C,F) Overlay images of embryos injected with in vitro transcribed GFP in fluorescence and DIC channels. Scale bar is 50 μm. Click here to view larger image.
Glass pasteur pipettes | VWR | 14673-043 | |
Inverted microscope Axiovert 40C | Zeiss | 4109431007990000 | Injection microscope |
Microloader tips | Eppendorf | 5242 956.003 | Load injection solution |
Nylon filter mesh 80 μm | Amazon.com | 03-80-37 | Filter eggs to get rid of debris |
P20 or P200 Aerosol Barrier Pipette Tips | Fisher Scientific | 02707432 or 02707430 | Part of a mouth pipette |
Parafilm | Fisher Scientific | 13 374 12 | Part of a mouth pipette |
Polyethylene tubing | Intramedic | PE-160 | Part of a mouth pipette |
Protamine sulfate | MP Biomedicals, LLC | 194729 | Attach dejellied eggs to injection dishes |
Sea urchins S. purpuratus | Pt. Loma Marine Invertebrate Lab | N/A | |
Sea water | any pet store | Instant Ocean | |
Sterile 60 mm x 15 mm Polystyrene Petri Dish | Fisher Scientific | 0875713A | Injection dishes |
Three-Axis Coarse Positioning Micromanipulator MMN-1 | Narishige | 9124 | Manipulate injection needle |
Three-Axis Joystick Type Oil Hydraulic Fine Micromanipulator MMO-202ND | Narishige | 9212 | Manipulate injection needle |
Transfer pipettes | Fisher Scientific | 13-711-9AM | |
Vertical needle puller | Narishige | PC-10 | Pull injection needles |
Microinjection into cells and embryos is a common technique that is used to study a wide range of biological processes. In this method a small amount of treatment solution is loaded into a microinjection needle that is used to physically inject individual immobilized cells or embryos. Despite the need for initial training to perform this procedure for high-throughput delivery, microinjection offers maximum efficiency and reproducible delivery of a wide variety of treatment solutions (including complex mixtures of samples) into cells, eggs or embryos. Applications to microinjections include delivery of DNA constructs, mRNAs, recombinant proteins, gain of function, and loss of function reagents. Fluorescent or colorimetric dye is added to the injected solution to enable instant visualization of efficient delivery as well as a tool for reliable normalization of the amount of the delivered solution. The described method enables microinjection of 100-400 sea urchin zygotes within 10-15 min.
Microinjection into cells and embryos is a common technique that is used to study a wide range of biological processes. In this method a small amount of treatment solution is loaded into a microinjection needle that is used to physically inject individual immobilized cells or embryos. Despite the need for initial training to perform this procedure for high-throughput delivery, microinjection offers maximum efficiency and reproducible delivery of a wide variety of treatment solutions (including complex mixtures of samples) into cells, eggs or embryos. Applications to microinjections include delivery of DNA constructs, mRNAs, recombinant proteins, gain of function, and loss of function reagents. Fluorescent or colorimetric dye is added to the injected solution to enable instant visualization of efficient delivery as well as a tool for reliable normalization of the amount of the delivered solution. The described method enables microinjection of 100-400 sea urchin zygotes within 10-15 min.
Microinjection into cells and embryos is a common technique that is used to study a wide range of biological processes. In this method a small amount of treatment solution is loaded into a microinjection needle that is used to physically inject individual immobilized cells or embryos. Despite the need for initial training to perform this procedure for high-throughput delivery, microinjection offers maximum efficiency and reproducible delivery of a wide variety of treatment solutions (including complex mixtures of samples) into cells, eggs or embryos. Applications to microinjections include delivery of DNA constructs, mRNAs, recombinant proteins, gain of function, and loss of function reagents. Fluorescent or colorimetric dye is added to the injected solution to enable instant visualization of efficient delivery as well as a tool for reliable normalization of the amount of the delivered solution. The described method enables microinjection of 100-400 sea urchin zygotes within 10-15 min.