Experimental design (Figures 2 and 3)
Choice of mouse strain. Embryos from the hybrid strain (C57/B6xDBA/2)F1 overcome the 2-cell block and develop into blastocysts in in vitro culture. This strain consistently produces at least 10-20 robust embryos per female. Mice should be 25-30 days old when superovulated to generate the most malleable embryos for injection. Also, pronuclei and polar bodies in these embryos are easy to identify, thus simplifying cytoplasmic injections.
Morpholinos. Morpholino sequence was designed according to manufacturer’s instructions. Morpholinos targeting Oct4 and controls are listed in Table 2.
Controls. Uninjected embryos serve as environmental condition negative controls for the MO-injected embryos. In addition, embryos injected with Human Globin MO (HG-MO) serve as negative controls to demonstrate that developmental hindrance is not an artifact of microinjection. In each experiment, uninjected embryos and embryos injected with HG-MO were tested in parallel with Oct4-MO-mediated knockdown. HG-MO was designed to specifically target a mutant human globin gene promoter, which would not be present in the mouse genome (Gene-Tools, Inc.). We had tested this HG-MO when establishing our methods and found that its presence did not affect blastocyst developmental rates. Of note, genes that were previously validated to be differentially-expressed between uninjected and Oct4-MO-injected embryos were also confirmed to show no differential expression between uninjected and HG-MO-injected embryos 21.
Injection station. Use of a Hoffman microscope with phase-contrast objectives and 40x magnification facilitates both the visual differentiation between an embryo and an oocyte, and the identification of pronuclei so they can be avoided during delivery of the MO into the cytoplasm. An anti-vibration platform (Visitek) placed directly under the microscope minimizes the possibility of trauma caused by a large puncture wound from a trembling needle. Tears from an unsteady needle can cause major damage to the embryo and may be the reason for the developmental arrest in some samples. Two micromanipulators attached to the microscope, one for holding and the other for injecting the embryo, move the glass pipettes in 3 different planes and are driven electrically and manually. Electrical control ensures fast coarse movement (bringing the needle/holding pipette into the microinjection drop), while the manual manipulator is used for the finer movements necessary to move and microinject embryos. Negative suction is controlled on the holding side by an Eppendorf Oil Tram, which creates negative/positive suction on the holding pipette. These glass holding pipettes were created with a pipette puller (Sutter Instrument P97) according to optimized conditions (Table 1) and finished on a microforge (De Fonbrune). The injection pipettes were also created on the Sutter’s P97 with program for injection pipettes/needle (Table 1).
Preparation and harvesting of preimplantation embryos (Figures 2, 3)
1 Superovulate 10 B6D2F1 females by intraperitoneal injection of 10 IU PMSG at 4-5 pm. Forty-eight hours later, inject the mice again with 10 IU hCG and mate each female with one male of the same strain 22. All procedures involving animals were performed under our active, Institutional Animal Care and Use Committee (IACUC) protocol #8315 entitled “Developmental Regulation of the Mammalian Embryo”, which was approved by the Administrative Panel on Laboratory Animal Care (APLAC) at Stanford University. TROUBLESHOOTING
2 Sacrifice the mice 17 h post-hCG injection by cervical dislocation, collect their oviducts, and place them in a 1.5 ml Eppendorf tube filled with M2 media. CRITICAL STEP We recommend that the adjacent uterine and ovarian tissue be dissected and removed en masse so that the embryos aren’t lost by damage to the oviduct 22. TROUBLESHOOTING
3 Under stereomicroscopic visualization, transfer all the oviducts into an organ dish filled with 1 ml warm M2 media. Using a Dumont #3 forceps, tease apart the ampullas and free the clusters of embryo-cumulus cells into the media. CRITICAL STEP If the ampullas are not easily located, then remove the zygotes by gently tearing apart the oviduct until embryos can be seen leaving through the open tears. Remove the oviduct and any residual tissue from the dish once the embryos are released.
4 Add 10 μl hyaluronidase stock solution to the organ dish and wait 5 minutes. As soon as embryos are freed from their clusters, remove them from the dish. Use a transfer pipette to place them into fresh wash droplets (~50 μL) of M2 in a 50 mm Petri dish.
5 Clean the embryos by pipetting them into several wash droplets. When all the follicular cumulus cells and debris have been thoroughly removed, place the embryos into a 35 mm petri dish filled with M2 and placed on a 37 °C warming plate.
Preparation of morpholinos (done concurrently with embryo harvest)
6 Resuspend the morpholino in sterile PCR grade water in certified DNase/RNase-free Eppendorf tubes. It is then heated in a thermocycler for 15 min at 65 °C to dissolve any precipitates that may clog the needle. TROUBLESHOOTING
7 During the 15-minute incubation period, pull fresh needles. When the MO is ready, load two needles and use immediately for microinjecting.
Intracytoplasmic morpholino microinjection
8 Place a chamber slide devoid of its chambers on the inverted microscope heating stage (set at 37 °C). Using a P1000 pipetteman, make a large dome shaped microinjection droplet (M2) on the chamber slide.
9 Using the micromanipulator coarse controls, lower the holding pipette and microinjection needle into the droplet. Orient the holding and microinjection pipettes on the left and right sides so that they are opposite one another in the field of view.
10 Turn on the microinjector air pressure. Once the injection needle is completely prepared, set the air pressure to 0.5-1.3 PSI so the MO is continuously flowing through the needle at a rate that will not lyse the embryo once it has been injected.
11 Transfer 40 embryos from the petri dish of M2 with a pipette into the microinjection droplet on the microscope stage. Reserve at least 15 of these embryos for the uninjected control group and sequester them from the batch of embryos meant for injection (Figure 2).
12 Proceed to inject 20-25 embryos in rapid succession. Hold each embryo so that the polar bodies are located either at the 6 or 12 o’clock position. Focus on the pronuclei by using the fine z-axis control. When injecting the embryo, avoid touching the pronuclei with the tip of the needle (Figure 4). At the end of an injection session, expect to see that up to 1/3 of injected embryos might not have survived injection. Carefully identify these defective embryos, and do not include them with injected or control embryos whose in vitro development will be observed.
13 Transfer the surviving injected embryos into a 50 μL drop of equilibrated culture medium (e.g., HTF/SS) overlaid with mineral oil in a 75 mm dish (the rinsing drops described in the Reagent Setup section). Wash these embryos in two more drops to remove residual M2 and transfer them to a culture dish. Culture 10 embryos per 20 μL drop of medium. Repeat this process with the uninjected embryos.
14 Place the culture dish in a glass dessicator and gas the chamber with Biomix for 1 minute. Put the dessicator into a 37 °C incubator and let the embryos recuperate for 2 hours before collecting them at the 1-cell stage, if that 1-cell stage embryos are required for downstream molecular analysis. CRITICAL STEP If not collecting cells at the 1-cell stage, do not open the incubator until the next day for observation and collection at the 2-cell stage.
RNA sample preparation for Affymetrix Gene Chip Analysis
15 Wash 20 embryos by transferring them through 3 drops of PBS/PVP. Deposit them into a 0.5 mL DNase/RNase-free tube and add 10 μL extraction buffer (XB from Picopure Total RNA Isolation Kit, Molecular Devices Corp.). Repeat for each set of embryos. Place embryos in a thermocycler and heat at 42 °C for 30 minutes. After heating, immediately store tubes at -80 °C for up to one month.
16 Proceed with extraction and isolation of total RNA after enough sets of pooled embryos have been collected (as instructed by the Picopure kit protocol). Each pool of 20 embryos should yield 10 μL of total RNA.
17 Use 5 μL of total RNA for two rounds of amplification (WT-Ovation Pico system, Nugen) per set of pooled embryos to yield 5-8 μg ssDNA. Process samples further with the FL-Ovation cDNA Biotin Module (Nugen).
Single Embryo RT-PCR
18 Transfer 5 embryos to a 50 μL drop of acid Tyrode’s solution to partially dissolve the zona pellucida. Wash these embryos by transferring them through 3 drops of PBS/PVP. Place each embryo in a 0.2 mL PCR strip tube and add 6.6 μL Sample RT-PreAmp Master Mix. Cap the tubes, vortex, quick spin, and place at -20 °C. Repeat with embryos for each condition.
19 Perform pre-amplification according to the protocol for direct mRNA gene quantification from individual cells using the Cells Direct Kit (Invitrogen), Taqman™ Gene Assays (Applied Biosystems) and perform semi-quantitative RT-qPCR to assay gene expression on the BioMark™ Dynamic Array (Fluidigm Corporation).