The Zebrafish Tol2 System: A Modular and Flexible Gateway-Based Transgenesis Approach

Transgenic zebrafish have proven critical to our understanding of embryo development. The Tol2 system is a versatile tool that enables researchers to quickly and systematically generate multiple transgenic zebrafish for FASD studies. The modular design and ease of generating new kit components make the Tol2 system a versatile tool for generating new transgenics without having to redesign any components of the kit.

My advice to researchers performing this technique for the first time is to make several trial runs to train on the precision of generating and injecting transgenic constructs. To begin, linearize the Tol2 kit plasmid containing transposase with a nought one restriction endonuclease by combining 10 microliters of transposase plasmid, 1.5 microliters of restriction endonuclease reaction buffer, and 0.3 microliters of nought one endonuclease in a 500 microliter microcentrifuge tube. Fill the reaction up to 20 microliters with RNAse-free water.

Mix and digest at 37 degrees Celsius overnight. The next day, stop the digestion by adding one microliter of 0.5 molar EDTA, two microliters of five molar ammonium acetate, and 80 microliters of 100%ethanol to the reaction mixture. Mix and chill at minus 20 degrees Celsius overnight.

The following day, centrifuge and aspirate the supernatant before resuspending the DNA pellet in RNAse-free water. Then determine the linear plasmid concentration in nanograms per microliter on a fluorometer by running two microliters of the sample. Set up the SP6 mRNA production by combining 10 microliters of 2X NTP/CAP, two microliters of 10X reaction buffer, 0.1 to one microgram of linear DNA template in two microliters of SP6 enzyme mix in a 500 microliter tube.

Fill the reaction up to 20 microliters with RNAse-free water and mix before incubating at 37 degrees Celsius. After two hours, add one microliter of DNAse, mix well, and incubate for an additional 15 minutes. Stop the reaction by adding 30 microliters of lithium chloride precipitation solution.

Mix and chill at minus 20 degrees Celsius overnight. Next day, centrifuge the solution to pellet the mRNA and aspirate the supernatant before washing the pellet with one milliliter of 80%ethanol. Air dry the pellet and resuspend it in 20 microliters of RNAse-free water.

Determine the mRNA concentration using a fluorometer, then aliquot 100 nanograms of mRNA per tube for single use and store at minus 80 degrees Celsius. To perform the reaction, gather 10 femtomole each of P5E, PME, and P3E and 20 femtomole of the destination vector PDEST. Identify the size of all four vectors and base pairs from the plasmid source.

After determining the concentration of all four vectors, using the weight of DNA as 660 grams per mole and plasmid size and base pairs, calculate the total nanograms of plasmid needed to reach either 10 femtomole for entry vectors or 20 femtomole for destination vector. Next, generate the construct sox17:EGFP-CAAX using the components described in the manuscript. Using the plasmid concentration, calculate the total microliters of each plasmid needed to reach either 10 femtomole or 20 femtomole, and then divide this by two for the use in the five microliters half LR reactions.

Generate 10 femtomole of P5E sox17, PME EGFP-CAAX, P3E Poly(A)and 20 femtomole of destination vector. Set up five microliters half LR reaction by combining the volumes of the P5E, PME, P3E, and PDEST in a 500 microliter tube. Then add sterile water to make the volume four microliters.

Vortex the LR enzyme mix twice for one minute each before adding one microliter to the reaction and mix thoroughly before incubating at 25 degrees Celsius. The following day, stop the LR reaction by adding 0.5 microliters of proteinase K.Incubate at 37 degrees Celsius for 10 minutes and then cool to room temperature. Next, transform three to four microliters of the plasmid into thawed chemically competent cells by adding the plasmid and letting the cells sit on ice for 25 to 30 minutes.

Then heat shock the cells in a 42 degree Celsius water bath for 30 seconds. After the heat shock, add 250 microliters of antibiotic-free rich liquid media to the cells and incubate with shaking at 37 degrees Celsius for 1.5 hours. Spread 300 microliters of bacterial suspension on one ampicillin Luria-Bertani plate and incubate overnight.

The following day, screen the colonies for the presence of two phenotypes:clear and opaque. Pick the clear colonies one at a time, inoculate the liquid culture, and shake at 37 degrees Celsius overnight. Combine 150 nanograms of plasmid, 100 nanograms of transposase mRNA, and 2%phenol red on ice.

Then add RNA-free water to make the volume three microliters. Transfer one cell stage embryo using transfer pipettes to the injection plate filled with EM completely covering the agar and then gently press approximately 50 to 75 embryos per slot of the injection plate. Add the mRNA plasmid phenol red mixture to the open end of a capillary injection needle.

Place the capillary needle in the injection rig armature and turn on the injection rig. Lower the needle into the injection plate and break the tip using forceps to allow only a small amount of mixture to be pumped out by the injection rig. Inject the embryos with a three nanoliter bolus of the mRNA plasmid phenol red mixture into the cell body of the embryo.

When finished, remove the embryos from the injection plate by gently popping them out of the slot and transfer pipetting them to a 100 millimeter Petri dish with fresh EM.Incubate at 28.5 degrees Celsius. Pick the appropriate developmental stage for fluorescent transgene expression and screen under a fluorescent dissecting microscope. Screen for non-fluorescent transgenes by screening for fluorescent transgenic markers such as GFP driven by the cardiac-specific promoter for the gene Cmlc2.

When the embryos positive for transgenic insertion develop into adults and reach the breeding age, screen them individually for germline transmission and transgene expressivity by breeding them to wild type zebrafish. Example bacterial colonies obtained from the transformation of the LR recombination are shown. Clear colonies contained a correct LR recombination product more than 85%of the time, whereas opaque colonies never contained a correct recombination product.

Diagnostic digestion of the three colonies from the transformation of the LR recombination products showed that the single opaque colony did not contain any plasmid, while the two clear colonies contained a single band at 9, 544 base pair. Transposase mRNA at 1, 950 base pair is shown here. Mosaic endoderm EGFP-CAAX expression was observed in 75%of the injected embryos.

Transgenic marker expression of Cmlc2 EGFP in the developing heart was observed 24 hours post-fertilization. Adult zebrafish had germline transmission of sox17:EGFP-CAAX generated fluorescent embryos with the endoderm fully labeled with EGFP-CAAX. Both the anterior-posterior length of the endoderm and the dorsal-ventral length of each pouch from pouches one to five was measured in control and ethanol-treated wild type BMP mutant embryos.

The overall length of the endoderm was not impacted by genotype or treatment. However, pouches one and three showed significant increases in pouch length between untreated and ethanol-treated wild type embryos, but significant decreases in length between untreated and ethanol-treated BMP mutants. Pouch two showed significant increases in pouch size between the untreated and ethanol-treated wild type and BMP mutant groups respectively.

When attempting this procedure, it is critical to be precise in pipetting and diluting of the Tol2 system, as well as hitting the cell body during embryo injection.