May 07, 2024

Public workspaceEfficient transfection protocol of rainbow trout gills epithelial (RTgill-W1) cell line through nucleofection system. V.2

DOI
dx.doi.org/10.17504/protocols.io.8epv5x4yng1b/v2
Efficient transfection protocol of rainbow trout gills epithelial (RTgill-W1) cell line through nucleofection system.
  • 1Pontificia Universidad Católica de Chile.;
  • 2Pontificia Universidad Catolica de Chile;
  • 3Universidad de Chile
  • Sebastián Escobar-Aguirre: Animal Science Department;
Sebastián Escobar-Aguirre
Open access
DOI: dx.doi.org/10.17504/protocols.io.8epv5x4yng1b/v2
External link: https://lbmm.cl/
Protocol Citation: Sebastián Escobar-Aguirre, Amanda Escorza, Matias Escobar-Aguirre 2024. Efficient transfection protocol of rainbow trout gills epithelial (RTgill-W1) cell line through nucleofection system.. protocols.io https://dx.doi.org/10.17504/protocols.io.8epv5x4yng1b/v2Version created by Sebastián Escobar-Aguirre
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: February 22, 2024
Last Modified: May 07, 2024
Protocol Integer ID: 99337
Keywords: Fish cell lines, RT gill, Electroporation, GFP
Funders Acknowledgement:
FONDECYT
Grant ID: 11190649
Abstract
In this protocol, we optimize the experimental condition to express plasmid DNA in salmonid fish rainbow trout gills epithelial (RTgill-W1), using a nucleofection system. The principle of nucleofection is a combination of electrical parameters that ensures efficient DNA delivery into the nucleus, combined with low toxicity and high cell viability. Using the Neon Transfection System (Invitrogen‱ MPK5000; code: 10431915) and the Invitrogen‱ Neon‱ Transfection System Kit 10 μl (brand: Invitrogen‱ MPK1096; code: 10124334) we were able to express over 60% of GFP in RTgill W 1 line cells. These results were achieved after trying different optimization steps voltage (ranges between 850 - 1600 volts), pulse exposure (times 10 30 ms), and number of pulses, being the condition 1600 volts, 20 ms 1 pulse, and 1 μg μL of plasmid the most effective compared to the other conditions, presenting a higher percentage of GFP expression and lower mortality rate.

Image Attribution
The image was taken by Matías Escobar-Aguirre, and corresponds to the RTgill-W1 image under a Cytation 5 microscope. Cell expressing GFP and staining the nucleus with Hoechst.

Materials
ReagentPBS - Phosphate-Buffered Saline (10X) pH 7.4Thermo Fisher ScientificCatalog #AM9625
ReagentTrypan Blue Solution 0.4% Sterile-filtered Merck MilliporeSigma (Sigma-Aldrich)Catalog #T8154
ReagentTrypsin EDTAGibco - Thermo FischerCatalog #25-051-CI.
ReagentLeibovitz's L-15 MediumThermo FisherCatalog #11415049
ReagentHoechst 33342, Trihydrochloride, Trihydrate - 10 mg/mL Solution in WaterThermo Fisher ScientificCatalog #H3570
Equipment
Neon Transfection system
NAME
electroporation
TYPE
Invitrogen
BRAND
MPK5000; code: 10431915
SKU

Equipment
Neon Transfection system
NAME
Pipette 10 ul
TYPE
Invitrogen
BRAND
MPK1096; code: 10124334)
SKU












Safety warnings
Attention
The protocol will be completely under a laminar flow hood and for the centrifugations the tubes will be closed in order to preserve sterility.
Before start
Cells were grown as a monolayer at 20 °C without CO 2 in Leibovitz L 15 medium (Cityva, USA) supplemented with 10 % heat-inactivated fetal bovine serum (FBS) (Biological Industries, Israel) and maintained at 1 x antibiotics solution ().
The culture must be at 70 - 80% confluency to ensure the exponential growth phase of the cells.

The electroporations will be using Amount10 µL tips and dispensed in Amount500 µL of fresh media in a 48- well plate.

Cell preparation
Cell preparation
1h
Before electroporation, cells should be between about 70 - 90% confluent (i.e 6x10e6 cells/ml in a 175 cm2 flask).
5m
Wash cells monolayer 2 times with Amount5 mL of 1X PBS and Amount1 mL of trypsin 0.25%- EDTA
10m
Neutralize cells with growth medium Amount9 mL into the flask.
5m
Take an aliquot of trypsinized cell suspension, and register the viable total cell number by trypan blue 1:1 or similar.
10m
From the total cell number (step 4), split 105 cells/well (48-well plates) in a new tube (i.e 4 reactions = 4 wells, we would have 4×105 cells).
5m
Critical
Centrifuge it Centrifigation500 x g, 4°C, 00:05:00 to pellet the cells.
5m
Remove the supernatant and wash withAmount5 mL of 1X PBS and repit step 6.
10m
Remove all PBS and resuspend the cells in Amount10 µL per well of Neon R buffer.
5m
Critical
Prepare a 48-well plate withAmount500 µL of medium WITHOUT ANTIBIOTIC where the cells will be seeded post-electroporation.

10m
Critical
NEON System Preparation
NEON System Preparation
10m
Prepare the Neon‱ electroporation tubes with Amount3 mL of electroporation buffer E and place it inside the Neon Pipette Station.
Add cells to the tube containing Amount1 undetermined of pEGFPC1 plasmid por well and gently mix. This plasmid encodes GFP (green fluorescent protein) as a reporter gene.
Mix
Insert the Neon tip into the Neon pipette (it is necessary to reach the second stop to open the clamp)
Electroporation
Electroporation
10m
Aspirate the cell/plasmidial DNA mix with the Neon tip. BUBBLES INSIDE THE TIP SHOULD BE AVOIDED AS THESE AFFECT THE VOLTAGE TRANSFER.
Pipetting
Critical
Insert the Neon pipette together with the Neon tip into the pipette station vertically until a click is heard
Apply 1600 volts for Duration00:20:00 and 1 pulse

20m
Critical
Carefully remove the Neon pipette from the station and immediately transfer the cells to a 48-well plate with medium WITHOUT ANTIBIOTIC and gently mix.
Culture in monolayer at 20 °C without CO2 in Leibovitz L 15 medium supplemented with 10% fetal bovine serum without antibiotic for 24 hours. Then change to fresh culture medium.
1d
Incubation
Optional
Determination of transfection efficiency
Determination of transfection efficiency
3d
Quantify/Estimate the number of live cells after 72 hours post electroporation.
3d
Determined transfection efficiency by measuring GFP fluorescence using the Cytation 5 platform from Agilent Technologies (excitation 469 emission 525).
It is recommended to use Hoechst staining (excitation 377 emission 447) to evaluate cell viability



Screening of cells electroporated 72 hours after electroporation with Hoechst solution. CHE−E cells correspond to cells that constitutively express GFP, as positive control.
Screening of cells electroporated 72 hours after electroporation with Hoechst solution. CHE−E cells correspond to cells that constitutively express GFP, as positive control.





Cells electroporated 72 hours after electroporation with Hoechst solution. A GFP stably expresses CHSE E cells used as GFP positive control. B Non-electroporated RTgill W 1 cells used as electroporation toxicity control. C RTgill W 1 cells electroporated using the conditions 1600 V, 20 ms and 1 pulse. D RTgill W 1 cells electroporated using the conditions 1500 V, 10 ms and 3 pulses
Cells electroporated 72 hours after electroporation with Hoechst solution. A GFP stably expresses CHSE E cells used as GFP positive control. B Non-electroporated RTgill W 1 cells used as electroporation toxicity control. C RTgill W 1 cells electroporated using the conditions 1600 V, 20 ms and 1 pulse. D RTgill W 1 cells electroporated using the conditions 1500 V, 10 ms and 3 pulses



Protocol references
We would like to thank Dr. Yehwa Jin for their help in setting up the condition.