Microinjection into the Caenorhabditis elegans embryo using an uncoated glass needle enables cell lineage visualization and reveals cell-non-autonomous adhesion control

Microinjection is a useful method in cell biology, with which exogenous substances are introduced into a cell in a location- and time-specific manner. The Caenorhabditis elegans embryo is an important model system for cell and developmental biology. Applying microinjection to the C. elegans embryo had been difficult due to the rigid eggshell surrounding the embryo. In 2013, microinjection method using a carbon-coated quartz needle for the C. elegans embryo was reported. To prepare the needle, unfortunately, special equipment is required and thus a limited number of researchers can use this method. In this study, we established a method for the microinjection of drugs, dyes, and microbeads into the C. elegans embryo using an uncoated glass needle that can be produced in a general laboratory. This method enabled us to easily detect cell lineage up to adult stages by injecting a fluorescent dye into a blastomere. We also found a cell-non-autonomous control mechanism of cell adhesion; specifically, the injection of an actin inhibitor into one cell at the 2-cell stage enhanced adhesion between daughter cells of the other cell. Our microinjection method is expected to be used for broad studies and could facilitate various discoveries using C. elegans.

4 DEVELOPMENTAL OUTCOMES AND EFFICIENCY OF TWO CRISPR/Cas9 MICROINJECTION METHODS IN BOVINE ZYGOTES

The CRISPR/Cas9 system is a fast, effective, and easy method for gene disruption, allowing generation of knockout animals by direct zygote injection. To date there is no report on the efficiency of this microinjection system in bovine zygotes and its effects on early development. The aim of this study was to compare 2 microinjection methods on developmental rates and efficiency to induce gene disruption. Microinjection effects on embryo development were evaluated by blastocyst (BL) formation rates at Day 8 of culture and by the proportion of lysed embryos (damaged during injection); while the efficiency of CRISPR/Cas9 RNA to create targeted mutations was studied by sequencing resulting blastocysts. Three groups were evaluated: (1) noninjected (control), (2) direct intracytoplasmic injection (direct-ICI), and (3) laser-assisted ICI (laser-ICI). Direct-ICI was performed with a beveled spiked glass needle (5 μm ID; Origio, Måløv, Denmark) to pass the zona pellucida (ZP) and deliver CRISPR/Cas9 RNA as earlier described (Ross et al. 2008 BMC Dev. Biol. 8, 16). For laser-ICI, a Research Instruments (RI) Saturn 5 Active™ laser system (Research Instruments Ltd., Falmouth, United Kingdom) was used to perforate the ZP, and a blunt-end glass needle (5–6 μm ID) used to deliver CRISPR/Cas9 RNA. In both cases, cytoplasm was aspirated into the pipette to disrupt the plasma membrane and the aspirated cytoplasm and CRISPR/Cas9 injected back into the embryo. Embryos were obtained by IVF of in vitro-matured oocytes aspirated from abattoir ovaries. At 18 h post-IVF (hpf), zygotes were denuded from cumulus cells and cultured in groups of 25 in 50-μL drops of KSOM (Evolve, Zenith Biotech, Guilford, CT, USA) with 4 mg mL–1 of BSA. Zygotes were injected at 20 to 22 hpf. Four biological replicates were assessed for BL rates (258 embryos total). A 2-tailed t-test was used to evaluate statistically significant differences (P ≤ 0.05) between groups. Direct-ICI had a greater proportion of lysed embryos (29.5 ± 10.6%) compared with laser-ICI (15.6 ± 5%; P = 0.056). BL development was significantly lower on direct-ICI (15.5 ± 8%) compared with laser-ICI (31.4 ± 5.9%; P = 0.02) and control groups (32.8 ± 6.6%; P = 0.01). These results indicate that laser-ICI causes less damage and results in normal BL rates after microinjection (P = 0.24). Because laser-ICI had normal BL rates and less embryo lysis, we used this method to evaluate efficiency of the CRISPR/Cas9 system to induce genomic mutations. Twelve BL were sequenced. Analysis of the CRISPR targeted region showed that 50% of the embryos had biallelic mutations, 33% monoallelic mutations, and 17% were wild type. These results show that laser-ICI method was very efficient for injecting CRISPR/Cas9 RNA into zygotes, resulting in normal developmental rates and a high amount of mutations in BL. The authors thank Research Instruments Ltd. for providing the laser used in this study.

TEM Sample Preparation Methods for MEMS Floating Structure Analysis

The MEMS structure has its particular character like hollow areas inside, and “floating” structures. Traditional TEM sample preparation method usually leads to distortion and dissociation defects of the floating structure. This paper introduces two innovative practical methods of TEM sample preparation using focused ion beam (FIB) for MEMS floating structure analysis. Method 1 used glass needle to lift out the separated film onto glue coated blank wafer; method 2 used in situ pick up system to lift out L- or C-shaped cut film onto TEM half-grid. And then the sample can be applied to normal TEM membrane preparation procedure.