An automated system for rapid cellular extraction from live zebrafish embryos and larvae: Development and application to genotyping.

Zebrafish are a valuable model organism in biomedical research. Their rapid development, ability to model human diseases, utility for testing genetic variants identified from next-generation sequencing, amenity to CRISPR mutagenesis, and potential for therapeutic compound screening, has led to their wide-spread adoption in diverse fields of study. However, their power for large-scale screens is limited by the absence of automated genotyping tools for live animals. This constrains potential drug screen options, limits analysis of embryonic and larval phenotypes, and requires raising additional animals to adulthood to ensure obtaining an animal of the desired genotype. Our objective was to develop an automated system that would rapidly obtain cells and DNA from zebrafish embryos and larvae for genotyping, and that would keep the animals alive. We describe the development, testing, and validation of a zebrafish embryonic genotyping device, termed "ZEG" (Zebrafish Embryo Genotyper). Using microfluidic harmonic oscillation of the animal on a roughened glass surface, the ZEG is able to obtain genetic material (cells and DNA) for use in genotyping, from 24 embryos or larvae simultaneously in less than 10 minutes. Loading and unloading of the ZEG is performed manually with a standard pipette tip or transfer pipette. The obtained genetic material is amplified by PCR and can be used for subsequent analysis including sequencing, gel electrophoresis, or high-resolution melt-analysis. Sensitivity of genotyping and survival of animals are both greater than 90%. There are no apparent effects on body morphology, development, or motor behavior tests. In summary, the ZEG device enables rapid genotyping of live zebrafish embryos and larvae, and animals are available for downstream applications, testing, or raising

Flow diagram.

<p>Flow diagram for genetic material extraction using the ZEG.</p

Behavior results following ZEG genotyping.

<p>Behavior results for 7 dpf larvae, comparing embryos genotyped on the ZEG to controls; there were no statistically significant differences. Box-plot analyses.</p

Testing of ZEG parameters.

<p>A) Evaluation for cross-contamination. Neighboring wells on a 24 channel chip were loaded either with an embryo or with a control (water blank); extraction was performed, followed by PCR and then HRMA. No signal was obtained in the water blank controls. Note, well B8 was designated in the software as a negative control and was therefore marked as a “Water (Negative)” with a block box; the maroon boxes labeled “Negative” were scored by the software algorithm as being negative/not amplifying. B) Representative images of cellular material collected following ZEG extraction; trypan blue staining. Scale bar 5 μm.</p

Views of glass slides (chips) for genotoyping.

<p>A) Schematic of glass slide, hydrophobic tape, and well design. B) 24 channel chip, top view; C) 24 channel chip loaded with 72 hpf zebrafish embryos; D) Base unit; E) Base unit with loading of 24 channel chip.</p

Compaction caused by mechanized operations in a Red- Yellow Latosol cultivated with coffee over time Compactação causada pelas operações mecanizadas em um Latossolo Vermelho-Amarelo cultivado com cafeeiros ao longo do tempo

The main source of soil structure degradation in coffee plantation is the machinery traffic because these operations may cause soil compaction affecting the crop development. This study aimed to generate the load-bearing capacity models for a Red-Yellow Latosol and to determine through the use of these models the soil susceptibility to compaction of the coffee plantation due to the implantation time and the compaction caused by the machinery traffic on the traffic lines located at the top and bottom of the ground. This study was carried out in the EPAMIG Experimental Farm, located at Três Pontas, MG, in coffee plantations (Coffee arabica L.) with 2, 7, 18 and 33 years of establishment. To obtain the load-bearing capacity models, 12 undisturbed soil samples were randomly collected in the 0-3 cm and 15-18 cm layers in the position between the rows for each establishment time of the coffee plantation. It was also randomly collected 10 undisturbed soil samples for each establishment time of the coffee plantations along the tractor traffic lines located at the top and bottom of the ground. These undisturbed soil samples were used in the uniaxial compression tests. The use of the load-bearing capacity models allow to identify the soil susceptibility to compaction due to the implementation time of the coffee plantation and the compaction caused by the machinery traffic on the traffic lines located at the top and bottom of the ground. The percentage of compacted soil samples increases with the establishment time in the layer of 15-18 cm.<br>A principal fonte de degradação da estrutura do solo na exploração cafeeira é o tráfego de máquinas, pois essas operações podem causar compactação do solo, afetando o desenvolvimento da cultura. Neste estudo, objetivou-se gerar os modelos de capacidade de suporte de carga para um Latossolo Vermelho-Amarelo e determinar por meio do uso desses modelos a suscetibilidade à compactação do solo, pelo tempo de implantação da lavoura cafeeira e os impactos causados pelo tráfego das operações mecanizadas nas linhas de tráfego localizadas na parte de cima e de baixo do terreno. Este estudo foi realizado na Fazenda Experimental da EPAMIG, situada em Três Pontas, MG, em lavouras cafeeiras (Coffee arabica L.) com 2, 7, 18 e 33 anos de implantação. Para a obtenção dos modelos de capacidade de suporte de carga, 12 amostras indeformadas de solo foram coletadas aleatoriamente nas camadas 0-3 e 15-18 cm no centro das entrelinhas, para cada tempo de implantação da lavoura cafeeira. Foram também coletadas aleatoriamente 10 amostras indeformadas para cada tempo de implantação da lavoura cafeeira ao longo das linhas de tráfego do trator situadas nas partes de cima e de baixo do terreno. Essas amostras foram usadas no ensaio de compressão uniaxial. O uso dos modelos de capacidade de suporte de carga permitiu identificar a suscetibilidade do solo à compactação, em razão do tempo de implantação da lavoura cafeeira e a compactação causada pelo tráfego do maquinário nas linhas de tráfego situadas na parte de cima e de baixo do terreno. A porcentagem de amostras compactadas aumentou com a idade da lavoura na camada de 15-18 cm