循環水槽

 このあいだ買った大水槽はこうなりました。

中にゼブラフィッシュ用の小型水槽を並べて簡易循環水槽にしました。

エーハイムの外部濾過フィルターから出た水をホームセンターで買ったアクリルパイプを通して各水槽に供給します。

9つの小型水槽を設置できました。

最近段々と飼育する魚が増えてきているのでこれで少し水槽のやりくりが楽になります。

水槽を買いました。

ゼブラフィッシュ飼育用の水槽を買いました。

特注です。東京アクアガーデンさんに作ってもらいました。

横 90cm ｘ 奥行 40cm ｘ 高さ 20cmです。

ここにそのまま魚を入れると水槽が大きすぎるので、これからちょっと工夫していきます。

Transfection of in vitro transcribed mRNAs in HEK293 cells

Transfection of mRNAs and FACS

Day 1 (cell seeding)

HEK293 cells (wild-type and PEX3 mutant clones) were seeded on a 6-well culture plate (TrueLine) 5 × 105 cell in 2mL / well and cultured at 37℃ 5% CO2 for overnight.

Day 2 (transfection)

The cells were transfected with PTS2-mCherry-PESTmRNA (the translated protein labels peroxisomes when they are present while immediately degrades when there is no or a few peroxisomes by PEST sequence) with EGFP mRNA (labels whole cell) using PEI MAX reagent (Polyscience) and Opti-MEM (ThermoFisherScientific). The cells were cultured for overnight.

Day 4 (FACS)

The cells were washed with PBS, dissociated by trypsin into single cells, and collected in a 15 mL tube with culture medium. After centrifugation (1000rpm 5min, )  cell pellets were resuspended in culture medium and proceed to FACS.

The cells were analyzed by a flow cytometer (Sony SH800). There are three populations in the cell such as an EGFP-positive and mCherry-high population, an EGFP-positive and mCherry-low population, and an EGFP-negative and mCherry-negative (untransfected) population.

The EGFP-positive mCherry-high population and the EGFP-positive mCherry-low population were separately collected by FACS. The collected cells were instantly frozen by liquid nitrogen and stored at negative 80℃ until LC-MS assay.

 

Transfection and single cell isolation

Day 1 (cell seeding)

PEX3 mutant clone #10 was seeded on a 6-well culture plate (TrueLine) 5 × 105 cell in 2mL / well and cultured at 37℃ 5% CO2 for overnight.

Day 2 (transfection)

The cells were transfected with mCherry-SKLmRNA (labels peroxisome when they are present in the cell while cytoplasm when absent or scarce) with EGFP mRNA (labels whole cell) using PEI MAX reagent (Polyscience) and Opti-MEM (ThermoFisherScientific). The cells were cultured at 37℃ 5% CO2 for overnight.

Day 3 (FACS)

The cells were washed with PBS, dissociated by trypsin into single cells, and collected in a 15 mL tube with culture medium. After centrifugation (1000rpm 5min, )  cell pellets were resuspended in culture medium and proceed to FACS.

The cells were analyzed by a flow cytometer (Sony SH800). Single cells of mCherry and EGFP positive cells were isolated in U-shape bottom 96-well plates (1 cell / well). Subsequently,  soritality and the peroxisomal phenotype of each cell was confirmed by a fluorescent microscope (Axioscope, Carl-Zeiss) and photographed (Axiocam, Carl-Zeiss). The cells were independently transferred to a flat-bottom 96-well culture plate and incubated for days to expand the clonal population. After the expansion, each cell clone was immunostained by anti-Catalase and anti-PMP70 antibodies to assess peroxisomal mosaicism.