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Size-Adjustable Microdroplets Generation Based on Microinjection

ABSTRACT: Microinjection is a promising tool for microdroplet generation, while the microinjection for microdroplets generation still remains a challenging issue due to the Laplace pressure at the micropipette opening. Here, we apply a simple and robust substrate-contacting microinjection method to microdroplet generation, presenting a size-adjustable microdroplets generation method based on a critical injection (CI) model. Firstly, the micropipette is adjusted to a preset injection pressure. Secondly, the micropipette is moved down to contact the substrate, then, the Laplace pressure in the droplet is no longer relevant and the liquid flows out in time. The liquid constantly flows out until the micropipette is lifted, ending the substrate-contacting situation, which results in the recovery of the Laplace pressure at the micropipette opening, and the liquid injection is terminated. We carry out five groups of experiments whereupon 1600 images are captured within each group and the microdroplet radius of each image is detected. Then we determine the relationship among microdroplet radius, radius at the micropipette opening, time, and pressure, and, two more experiments are conducted to verify the relationship. To verify the effectiveness of the substrate-contacting method and the relationship, we conducted two experiments with six desired microdroplet radii are set in each experiment, by adjusting the injection time with a given pressure, and adjusting the injection pressure with a given time. Then, six arrays of microdroplets are obtained in each experiment. The results of the experiments show that the standard errors of the microdroplet radii are less than 2% and the experimental errors fall in the range of ±5%. The average operating speed is 20 microdroplets/min and the minimum radius of the microdroplets is 25 ?m. This method has a simple experimental setup that enables easy manipulation and lower cost.

SUBMITTER: Li S

PROVIDER: S-EPMC6189902 | biostudies-literature | 2017 Mar

REPOSITORIES: biostudies-literature

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Project description:While CRISPR/Cas9 technology has proven to be a valuable system to generate gene-targeted modified animals in several species, this tool has been scarcely reported in farm animals. Myostatin is encoded by MSTN gene involved in the inhibition of muscle differentiation and growth. We determined the efficiency of the CRISPR/Cas9 system to edit MSTN in sheep and generate knock-out (KO) animals with the aim to promote muscle development and body growth. We generated CRISPR/Cas9 mRNAs specific for ovine MSTN and microinjected them into the cytoplasm of ovine zygotes. When embryo development of CRISPR/Cas9 microinjected zygotes (n = 216) was compared with buffer injected embryos (n = 183) and non microinjected embryos (n = 173), cleavage rate was lower for both microinjected groups (P<0.05) and neither was affected by CRISPR/Cas9 content in the injected medium. Embryo development to blastocyst was not affected by microinjection and was similar among the experimental groups. From 20 embryos analyzed by Sanger sequencing, ten were mutant (heterozygous or mosaic; 50% efficiency). To obtain live MSTN KO lambs, 53 blastocysts produced after zygote CRISPR/Cas9 microinjection were transferred to 29 recipient females resulting in 65.5% (19/29) of pregnant ewes and 41.5% (22/53) of newborns. From 22 born lambs analyzed by T7EI and Sanger sequencing, ten showed indel mutations at MSTN gene. Eight showed mutations in both alleles and five of them were homozygous for indels generating out-of frame mutations that resulted in premature stop codons. Western blot analysis of homozygous KO founders confirmed the absence of myostatin, showing heavier body weight than wild type counterparts. In conclusion, our results demonstrate that CRISPR/Cas9 system was a very efficient tool to generate gene KO sheep. This technology is quick and easy to perform and less expensive than previous techniques, and can be applied to obtain genetically modified animal models of interest for biomedicine and livestock.

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Size-Adjustable Microdroplets Generation Based on Microinjection

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