Methods for Precisely Localized Transfer of Cells or DNA into Early Postimplantation Mouse Embryos

The overall goal of these procedures is to demonstrate how to precisely transfer cells or DNA into early post implantation mouse embryos. These methods can help answer key questions in developmental biology, such as testing the in vivo potential of in vitro cultured cells and the functions of certain genes in specific embryonic stages. The main advantages of these techniques are that they do not require any specialized equipment and render experimental manipulations of early posing plantation mouse bryo possible After sacrificing a pregnant female on day E 7.5 or E 8.5 according to the text protocol, use scissors and forceps to isolate the uterus and place it in a 30 millimeter dish filled with M two medium with two pairs of fine forceps.

Carefully tear apart the myometrium, then peel away the decidua, being careful not to puncture the extra embryonic cavities. Remove the riker's membrane by using forceps to pinch it and slowly separate it from the embryo. Then under a stereo dissecting microscope, check the embryos to ensure that the yolk sac amnion and op placental cone are intact With a pipette, transfer the embryos to a clean dish of M two and place on a 30 millimeter plastic Petri dish lid on ice or an ice platform to partially chill the embryos.

Prepare culture medium and culture embryos according to the text protocol to graft cultured cells into mouse embryos. Begin by using a 200 microliter pipette tip to physically scrape eblast stem cells, which ubiquitously express GFP from a six well culture plate. Transfer the cells into the dish containing the embryos.

Attach a hand pulled grafting capillary to the aspirator tube to make a mouth pipette gently suck the mouth pipette to draw one or more cell clumps of greater than 20 cells into the grafting capillary. Then gently blow out the cells to disperse large clumps. Select one clump containing about 10 to 20 cells and draw it into the grafting capillary.

Again, keeping the clump close to the opening of the capillary with a pair of forceps. Hold the embryo loosely in place and insert the grafting capillary into the region of interest. To create an opening.

Gently expel the clump out of the grafting capillary, leaving the short string of 10 to 20 cells lodged in the embryo. Leave the embryos in the same dish of M two medium and use a fluorescence compound dissecting microscope and camera to image the grafted embryos. Keep the imaging time to a minimum to avoid exposure of the embryos to excessive light and heat.

Immediately after imaging using a paste, transfer the embryos with a minimal volume of M two medium to pre equilibrated culture medium and culture according to the guidelines in the text protocol prior to carrying out electroporation experiments, use a horizontal micro pipette polar to pull DNA injection pipettes with a fine tip and an opening of less than 10 micrometers To avoid tissue damage. For glass capillary electroporation, use a micro forge to cut the opening of the DNA injection pipettes to an internal diameter of either 20 or 30 micrometers with a clean tip and no broken edges. Attach each platinum electrode to a thin insulated wire and insert it into a microinjection needle holder covered with insulation tape.

To prepare a handmade capillary electrode insert a 0.2 millimeter diameter platinum wire into an electroporation glass capillary with a fixed opening of 20 or 30 micrometers in diameter. To focus the electric current and deliver the plasma DNA to a small region of interest in the embryo. To make an L-shaped electrode bend a 0.2 millimeter diameter platinum wire, creating an L shape with the horizontal portion of the L around one millimeter in length.

Mount the needle holders on standard micro-manipulation instrument holders. Connect the capillary electrode to the anode of the power supply. Connect the L-shaped electrode to the anode of the multimeter.

Then connect the cathode of the multimeter to the cathode of the power supply. Fill the electroporation glass capillary with PBS to within one to two millimeters of the top and insert the straight platinum electrode into the glass capillary until it reaches the bottom of the capillary. Anchor the L-shaped electrode on the surface of the 30 millimeter petri dish filled with PBS.

Use a pneumatic pico pump for DNA injection. Insert the injection needle from the lateral eblast into the amniotic cavity of the embryo and inject approximately five microliters of DNA solution into the cavity or until it is completely full. Take care not to burst the embryo.

Then carefully position the embryo between the electrodes and move the capillary electrode to the precise location where the DNA is to be delivered. Using 200 volts in six pulses each 50 milliseconds in duration, electroporated the embryo with a one second interval between each pulse. Immediately transfer the embryo to pre equilibrated culture medium before repeating the electroporation for the next embryo.

Detect electroporated live or dead cells two hours after electroporation. According to the text protocol shown here is an embryo with epi PSCs ubiquitously expressing EGFP grafted on E 7.5 and cultured ex vivo for 24 hours. When 10 to 16 fscs were grafted into E 7.5 embryos, they incorporated and proliferated well within the host embryo.

However, grafting more cells does not result in better chimerism and in fact results in unincorporated clumps as demonstrated here. As shown in this electroporation of GFP expressing plasmids. GFP positive cells were detected one to two hours after electroporation.

When distal eblast cells in the late primitive streak stage embryo were electroporated labeled cells contributed to the neural derm after 24 hours in culture, which corresponds to known fate maps of eblast cells in gastro stage embryos. This figure shows that similar to using other types of electrodes for electroporation, the capillary electrode also caused some degree of cell death in the targeted region. As this region appeared darker in color compared to neighboring regions labeling the nuclei of dead cells with a membrane impermeable far red fluorescence die.

Confirmed that the capillary electroporation technique results in only a small number of dead cells near the electroporation site. While attempting these procedures, it is important to start culturing the embryos within two hours. After isolating the uterine from the mice at the end of the culture, the embryos can be fixed.

Other methods like sectioning staining can be performed in order to answer additional questions like the contribution of a donor or electro braided cells to the host embryos. After watching this video, we should have a good understanding of how to precisely transfer cells our DNA into early post implantation mouse embryos.