This work describes a protocol for the modular Tol2 transgenesis system, a gateway-based cloning method to create and inject transgenic constructs into zebrafish embryos.
Fetal alcohol spectrum disorders (FASD) are characterized by a highly variable set of structural defects and cognitive impairments that arise due to prenatal ethanol exposure. Due to the complex pathology of FASD, animal models have proven critical to our current understanding of ethanol-induced developmental defects. Zebrafish have proven to be a powerful model to examine ethanol-induced developmental defects due to the high degree of conservation of both genetics and development between zebrafish and humans. As a model system, zebrafish possess many attributes that make them ideal for developmental studies, including large numbers of externally fertilized embryos that are genetically tractable and translucent. This allows researchers to precisely control the timing and dosage of ethanol exposure in multiple genetic contexts. One important genetic tool available in zebrafish is transgenesis. However, generating transgenic constructs and establishing transgenic lines can be complex and difficult. To address this issue, zebrafish researchers have established the transposon-based Tol2 transgenesis system. This modular system uses a multisite Gateway cloning approach for the quick assembly of complete Tol2 transposon-based transgenic constructs. Here, we describe the flexible Tol2 system toolbox and a protocol for generating transgenic constructs ready for zebrafish transgenesis and their use in ethanol studies.
Prenatal ethanol exposure gives rise to a continuum of structural deficits and cognitive impairments termed fetal alcohol spectrum disorders (FASD)1,2,3,4. The complex relationships between multiple factors make studying and understanding the etiology of FASD in humans challenging. To resolve this challenge, a wide variety of animal models have been used. The biological and experimental tools available in these models have proven crucial in developing our understanding of the mechanistic basis of ethanol teratogenicity, and the results from these model systems have been remarkably consistent with what is found in human ethanol studies5,6. Among these, zebrafish have emerged as a powerful model to study ethanol teratogenesis7,8, in part due to their external fertilization, high fecundity, genetic tractability, and translucent embryos. These strengths combine to make zebrafish ideal for real-time live imaging studies of FASD using transgenic zebrafish lines.
Transgenic zebrafish have been extensively used to study multiple aspects of embryonic development9. However, creating transgenic constructs and subsequent transgenic lines can be exceedingly difficult. A standard transgene requires an active promoter element to drive the transgene and a poly A signal or "tail", all in a stable bacterial vector for general vector maintenance. The traditional generation of a multi-component transgenic construct requires multiple time-consuming sub-cloning steps10. PCR-based approaches, such as Gibson assembly, can circumvent some of the issues associated with sub-cloning. However, unique primers must be designed and tested for the generation of every unique transgenic construct10. Beyond transgene construction, genomic integration, germline transmission, and screening for proper transgene integration have been difficult as well. Here, we describe a protocol for using the transposon-based Tol2 transgenesis system (Tol2Kit)10,11. This modular system uses multisite Gateway cloning to quickly generate multiple transgenic constructs from an ever-expanding library of "entry" and "destination" vectors. Integrated Tol2 transposable elements greatly increase the rate of transgenesis, allowing for the rapid construction and genomic integration of multiple transgenes. Using this system, we show how the generation of an endoderm transgenic zebrafish line can be used to study the tissue-specific structural defects underlying FASD. Ultimately, in this protocol, we show that the modular setup and the construction of transgenic constructs will greatly aid zebrafish-based FASD research.
All zebrafish embryos used in this procedure were raised and bred following established IACUC protocols12. These protocols were approved by the University of Louisville.
NOTE: The wild-type zebrafish strain, AB, and the bmp4st72;smad5b1100 double mutant line were used in this study. All the water used in this procedure was sterile reverse osmosis water. Confocal images were taken under a laser-scanning confocal microscope. The endoderm measurements were made using the measure tool in ImageJ. All the statistical analyses were performed using statistical software.
1. Making the solutions and media
2. Embryo injection molds
3. Injection pipettes
4. Transposase mRNA preparation
5. Multisite Gateway cloning to create entry vectors for transgenesis
NOTE: This protocol is modified from Kwan et al.10, with the LR reaction written as a half-LR reaction and with a total volume of 5 µL. To generate new entry elements, BP reactions using 5', middle, and 3' donor vectors need to be used10,13.
6. Injection of the transgene into the embryos
7. Screening embryos for transgenic insertion
To generate the transgenic constructs, we used the Tol2 transgenesis system. Three entry vectors, including p5E, which holds the gene promoter/enhancer elements, pME, which holds the gene to be expressed by the promoter/enhancer elements, and p3E which, at minimum, holds the polyA tail, were used to generate the transgenic construct via multisite gateway LR cloning. The destination vector, pDest, provides the Tol2 repeats for the genomic insertion of the transgenic construct in zebrafish embryos and contains all the essential genetic information for bacterial growth (Figure 1A). For the purposes of this manuscript, we created and injected a sox17:EGFPCAAX transgenic construct. The promoter of the endoderm marker, sox17, was used to drive membrane-tagged EGFP in the developing endoderm of the zebrafish. For transgenic constructs that express non-fluorophore proteins, a pDest vector that contains a fluorescent transgenic marker such as cmlc2:EGFP can be used to aid in genomic insertion (Figure 1A).
Using the values described in the protocol above (Table 2), the sox17:EGFPCAAX transgenic construct was generated, and 4 µL of this construct was transformed into chemically competent Escherichia coli cells. After incubating at 37 °C overnight, the agar plate contained approximately 250 colonies (LR reactions typically average 150-300 colonies per plate in our lab). The screening of these colonies was performed based on opacity. In our hands, colonies that were clear had the correct sox17:EGFPCAAX product >85% of the time, whereas the opaque colonies never contained the correct recombination product (Figure 1B, arrowhead vs. arrow). After isolating the plasmid from several colonies, the restriction digestion of the recombinant products was performed with the restriction enzyme, NcoI. Two different clear colonies and one opaque colony were digested. Both clear colonies had a single band at the correct size of 9,544 bp (undigested plasmids loaded as a digest control), while the opaque colony did not have any bands at all (Figure 2A). These positive sox17:EGFPCAAX constructs were retransformed, the subsequent colonies were re-streaked, those colonies were confirmed to have the plasmid, and −80 °C bacterial freezer stocks were generated. The concentrations of both the sox17:EGFPCAAX plasmid and the capped-transposase mRNA were determined so they could both be used for injection (Figure 2B).
The embryos were prepared for injection by placing one cell-stage embryos into a pre-warmed embryo injection mold (Figure 3A–C). Once placed in the injection mold, the injection needle containing the sox17:EGFPCAAX mRNA was placed in a micropipette holder on the micromanipulator (Figure 3D,E). The embryos were injected with 100 ng of transposase mRNA, 150 ng of sox17:EGFPCAAX plasmid, and phenol red (injection tracking dye). A 3 nL bolus (determined by size as described in the protocol, step 6.6) was injected into the cell body and not into the yolk of the embryo for the best chance of integration (Figure 3F)10.
After injection, the embryos were removed from the injection mold and allowed to develop for 24 h. At 24 h post fertilization (hpf), the embryos were screened for the endoderm expression of EGFPCAAX. Mosaic endoderm EGFPCAAX expression was observed in ~75% of the injected embryos (Figure 4A,A'). To screen the embryos injected with non-fluorescent transgenes such as Gal4 or CreERT2, a destination vector that also contains a transgenic marker (i.e., cmlc2:EGFP) (Figure 1A) can be used (Figure 4B,B'). Adult zebrafish that had germline transgenesis of sox17:EGFPCAAX generated fluorescent embryos with the endoderm fully labeled with EGFPCAAX (Figure 4C).
Using the newly created sox17:EGFPCAAX transgenic line, we were able to directly assess the impact of ethanol on the formation of the endodermal pouches. The pouches are protrusions that form on the lateral edge of the endoderm and are important for the formation of the facial skeleton and multiple organ systems17. We have previously shown that blocking bone morphogenetic protein (Bmp) signaling results in hypoplasia of the pouches18. We now show that the ethanol treatment of Bmp mutants from 10-18 hpf has a subtle yet significant impact on pouch size. The dorsal-ventral length of each pouch from pouches 1-5 (zebrafish have six pouches, but the sixth had yet to fully form at the developmental stage imaged) was measured in control and ethanol-treated wild-type Bmp mutant embryos (Figure 5A). As a control for general growth impacts due to ethanol exposure, the anterior-posterior length of the entire endoderm was measured (Figure 5A). The overall length of the endoderm was not impacted by genotype or treatment (Figure 5B). However, pouches 1 and 3 showed significant increases in pouch length between the untreated and ethanol-treated wild-type embryos but significant decreases in length in between the untreated and ethanol-treated Bmp mutants (Figure 5C; two-way ANOVA; pouch 1: F(1,54) = 10.39, p = 0.0021; pouch 3: F(1,54) = 12.70, p = 0.0008). Pouch 2 showed significant increases in pouch size between the untreated and ethanol-treated wild-type and Bmp mutant groups, respectively (Figure 5C; two-way ANOVA; F(1,54) = 18.94, p < 0.0001).
Figure 1: LR gateway reaction for transgene construction. (A) Schematic of the modular four-part LR gateway cloning reaction. LR Clonase recombines the three entry vectors, p5E, pME, and p3E, and the destination vector, pDest, in a highly specific reaction to generate a novel transgenic product ready for embryo injection. For screening non-fluorescent transgenes, pDest vectors can contain optional transgenic markers (cmlc2:EGFP, as an example). (B) Example bacterial colonies obtained from the transformation of the LR recombination products. Clear colonies contained a correct LR recombination product >85% of the time (arrowhead), whereas opaque colonies never contained a correct recombination product (arrow). Please click here to view a larger version of this figure.
Figure 2: Analysis of the plasmid DNA and transposase mRNA. (A) Diagnostic digestion of the three colonies from the transformation of the LR recombination products. The single opaque colony did not contain any plasmid to screen, while the two clear colonies contained a single band at 9,544 bp (uncut vs. cut). (B) Transposase mRNA at 1,950 bp. Please click here to view a larger version of this figure.
Figure 3: Zebrafish embryo injection setup. (A) An injection mold is placed in liquid agarose diluted in EM (3% v/v). (B) Once solidified, the mold is removed from the agarose plate. (C) The embryos are arranged in the injection molds. (D,E) The mRNA/plasmid/phenol red mixture is pipetted into the injection needle, which is placed in the micropipette holder in the micromanipulator. (F) A 3 nL bolus is injected into the cell body of the embryo at the one-cell stage. Please click here to view a larger version of this figure.
Figure 4: Screening zebrafish injected with the transgenic construct. (A,A') Confocal image of an embryo imaged at 24 hpf shows the mosaic expression of the sox17:EGFPCAAX transgene. (B,B') Transgenic marker expression of cmlc2:EGFP in the developing heart at 24 hpf. Lateral views of the embryos, with anterior to the left and dorsal at the top. (C) Confocal image of an embryo generated from a transgene-carrying adult zebrafish. The entire endoderm is expressing EGFPCAAX. Lateral view of the embryo, with anterior to the left and ventral at the top. Please click here to view a larger version of this figure.
Figure 5: Pouch measures in ethanol-treated wild-type and Bmp mutant embryos. (A) Schematic showing the measurement of the overall length of the endoderm and the length of the pouches. (B) The measurements of the overall anterior-posterior endoderm length show no difference between untreated and ethanol-treated wild-type and Bmp mutant embryos. (C) The measurements of dorsal-ventral pouch length indicate that pouches 1 and 3 show increases in pouch length between the untreated and ethanol-treated wild-type embryos but decreases in length between the untreated and ethanol-treated Bmp mutants (two-way ANOVA; pouch 1: F(1,54) = 10.39, p = 0.0021; pouch 3: F(1,54) = 12.70, p = 0.0008). Pouch 2 shows an increase in length between the untreated and ethanol-treated wild-type and Bmp mutant groups (two-way ANOVA; F(1,54) = 18.94, p < 0.0001). No differences were observed in pouch length between pouches 4 and 5. Please click here to view a larger version of this figure.
Table 1: Tol2Kit components housed in the Lovely Lab. Every entry and destination vector available in the Lovely Lab, their descriptions, and their lab of origin. Please click here to download this Table.
Table 2: Calculation of the amount and concertation of each vector in the LR recombination reaction. The amount of each vector was calculated from the size (in bp) and the fmol needed for each component: 10 fmol of each entry vector and 20 fmol of the destination vector. From the plasmid concentration, each vector is diluted in sterile water to add 1 µL or less to the LR reaction. Sterile water is added to the vector pool to equal 4 µL, 1 µL of LR reaction mix is added to the reaction, and it is incubated at 25°C overnight. Please click here to download this Table.
Zebrafish are ideally suited for studying the impact of ethanol exposure on development and disease states7,8. Zebrafish produce large numbers of translucent, externally fertilized, genetically tractable embryos, which allows for the live imaging of several transgene-labeled tissues and cell types simultaneously in multiple environmental contexts19,20. These strengths, combined with the strong developmental genetic conservation with humans, make zebrafish a powerful model system for imaging the impact of ethanol7,8. Here, we described the protocol for a modular approach to generating and injecting transgenic constructs and establishing transgenic zebrafish lines for future ethanol studies.
Many different approaches have been established to generate transgenic zebrafish. However, generating both transgenic constructs and transgenic lines can be daunting. While traditional sub-cloning techniques, BAC cloning, or PCR-based approaches such as Gibson assembly allow for generating transgenic constructs, they have low throughput and lack versatility in their construction10. In addition, these strategies need to be redesigned for every transgenic construct created. Sub-cloning requires multiple digestions and ligations, assuming all the restriction sites necessary are present and unique. BAC cloning requires the sequencing and testing of multiple BAC clones. For Gibson assembly, new PCR primers have to be designed for each transgenic construct. Furthermore, the injection of either uncut or linearized DNA leads to low germline transmission21,22,23.
The Tol2Kit is a modular system that uses gateway cloning to generate complete transgenic constructs flanked by Tol2 repeats that, when combined with transposase mRNA, greatly increase transgenesis and germline transmission10,11,24,25. Dozens of entry and destination vectors are available from the Kwan lab and the Cole Lab10,11. In addition, zebrafish labs that use the Tol2Kit have generated and curated many more entry and destination vectors. As an example of the breadth of vectors available, we have pooled and used over 70 vectors (Table 1). With all these community resources, one can quickly generate thousands of different transgenic constructs by simply combining the vectors of choice in an LR reaction.
Optimal LR reactions do require exact calculations of plasmid size and concentration. The calculations for each vector are done in femtomole (fmol) values, so each reaction does not require a high plasmid volume to generate a transgenic construct. However, this small amount of plasmid makes dilutions and proper pipetting exceedingly critical to the success of the reaction10. Additional factors that can impact the LR reaction are the size of the inserts in the different entry vectors. For example, the p5E-sox17 element used in this study is over 4 kb, which, if combined with equally large pME and p3E elements, will result in a very large transgenic vector. A large plasmid in bacteria can be difficult to culture, thus decreasing the number of colonies generated from the bacterial transformation. This will also result in slower growth and decreased plasmid levels when isolated10. Importantly, having highly competent bacterial cells, as well as increasing the incubation of the bacterial cells during the transformation of the recombination plasmid, are key to generating enough colonies to capture proper transgene formation. In addition, using the correct LR reaction enzyme (listed in the Table of Materials) also plays a major role in the success of the LR reaction.
Beyond the generation of a transgenic construct and bacterial transformations, the embryo injection and genome integration can be difficult as well. The generation of high-quality transposase mRNA greatly increases the frequency of genomic integration10. The pulled capillaries need to be made just shortly before injecting the embryos as older needles can lose capillary action (i.e., the injection fluid fails to move to the tip of the needle) (Figure 3E). Both breaking the tip of the needle to only inject ~3 nL of fluid and injecting the cell body require practice. Our training protocol involves injecting the cell body with only phenol red injection dye until breaking the needle and injecting the embryos are mastered. Injecting the cell body drastically increases the rate of genome integration10. Combining all this, we average 75% or greater genome insertion and mosaic expression, but this can vary from construct to construct.
Since genomic insertion can be random, every embryo that shows mosaic expression is a unique transgenic insertion and requires continued screening. This continued screening is necessary to show that the integration site is not detrimental to the development of the embryo, that germline transmission occurs, and that the expression of the transgene is not attenuated or potentially silenced. Once a transgenic line has been established, it can be readily used for multiple analyses in ethanol studies. For those non-fluorescent transgenic constructs, commonly used transgenic markers include cmlc2:GFP and αcrystallin:RFP, which label the heart and retina, respectively, and allow for continued transgenic screening. In addition to transgenic markers for screening, these two transgenes can be used to directly study the impact of ethanol on heart and retina development.
Using the protocol described above, we generated a sox17:EGFPCAAX transgenic zebrafish line that had stable germline transmission of an endoderm-specific membrane-tagged EGFP construct. Using this line, we were able to measure the impact of ethanol on endodermal pouch formation in ethanol-sensitive Bmp mutant embryos. We showed that the sizes of pouches 1-3 but not of pouches 4 and 5 nor the overall endoderm length were impacted in ethanol-treated Bmp mutants (Fig 5). This pilot work suggests that the Bmp-ethanol interaction disrupts endoderm formation, in particular the cell behaviors underlying pouch formation. This work exemplifies the utility of generating transgenic zebrafish lines for ethanol studies. As a result, creating novel transgenic lines will greatly increase our understanding of ethanol-sensitive cellular processes and tissue events in FASD.
Ultimately, transgenic zebrafish, and zebrafish in general, have proven to be incredibly powerful in the study of FASD7,8. The Tol2Kit is an extremely versatile toolkit that enables researchers to quickly generate multiple transgenic constructs ready to inject into zebrafish. The modular design and the ease of generating new entry vectors result in extreme flexibility in generating transgenic constructs without having to redesign any components. Overall, this toolkit will greatly improve both zebrafish research and research in general aimed at improving the understanding of FASD.
The authors have nothing to disclose.
The research presented in this article was supported by a grant from the National Institutes of Health/National Institute on Alcohol Abuse (NIH/NIAAA) R00AA023560 to C.B.L.
Addgene Tol2 toolbox | https://www.addgene.org/kits/cole-tol2-neuro-toolbox/ | ||
Air | Provided directly by the university | ||
Ampicillin | Fisher Scientific | BP1760 | |
Analytical Balance | VWR | 10204-962 | |
Borosil 1.0 mm OD x 0.75 mm ID Capillary | FHC | 30-30-0 | |
Calcium Chloride | VWR | 97062-590 | |
Chloramphenicol | BioVision | 2486 | |
EDTA | Fisher Scientific | BP118-500 | |
Fluorescent Dissecting Microscope | Olympus | SZX16 | |
Kanamycin | Fisher Scientific | BP906 | |
Laser Scanning Confocal Microscope | Olympus | Fluoview FV1000 | |
Lawsone Lab Donor Plasmid Prep | https://www.umassmed.edu/lawson-lab/reagents/lawson-lab-protocols/ | ||
LB Agar | Fisher Scientific | BP9724 | |
LB Broth | Fisher Scientific | BP1426 | |
Low-EEO/Multi-Purpose/Molecular Biology Grade Agarose | Fisher Scientific | BP160-500 | |
LR Clonase II Plus Enzyme | Fisher Scientific | 12538200 | |
Magnesium Sulfate (Heptahydrate) | Fisher Scientific | M63-500 | |
Micro Pipette holder | Applied Scientific Instrumentation | MIMPH-M-PIP | |
Microcentrifuge tube 0.5 mL | VWR | 10025-724 | |
Microcentrifuge tube 1.5 mL | VWR | 10025-716 | |
Micromanipulator | Applied Scientific Instrumentation | MM33 | |
Micropipette tips 10 μL | Fisher Scientific | 13611106 | |
Micropipette tips 1000 μL | Fisher Scientific | 13611127 | |
Micropipette tips 200 μL | Fisher Scientific | 13611112 | |
mMESSAGE mMACHINE SP6 Transcription Kit | Fisher Scientific | AM1340 | |
Mosimann Lab Tol2 Calculation Worksheet | https://www.protocols.io/view/multisite-gateway-calculations-excel-spreadsheet-8epv599p4g1b/v1 | ||
NanoDrop Spectrophotometer | NanoDrop | ND-1000 | |
NcoI | NEB | R0189S | |
NotI | NEB | R0189S | |
Petri dishes 100 mm | Fisher Scientific | FB012924 | |
Phenol Red sodium salt | Sigma Aldrich | P4758-5G | |
Pipetman L p1000L Micropipette | Gilson | FA10006M | |
Pipetman L p200L Micropipette | Gilson | FA10005M | |
Pipetman L p2L Micropipette | Gilson | FA10001M | |
Potassium Chloride | Fisher Scientific | P217-500 | |
Potassium Phosphate (Dibasic) | VWR | BDH9266-500G | |
Pressure Injector | Applied Scientific Instrumentation | MPPI-3 | |
QIAprep Spin Miniprep Kit | Qiagen | 27106 | |
Sodium Bicarbonate | VWR | BDH9280-500G | |
Sodium Chloride | Fisher Scientific | S271-500 | |
Sodium Phosphate (Dibasic) | Fisher Scientific | S374-500 | |
Stericup .22 µm vacuum filtration system | Millipore | SCGPU11RE | |
Tol2 Wiki Page | http://tol2kit.genetics.utah.edu/index.php/Main_Page | ||
Top10 Chemically Competent E. coli | Fisher Scientific | C404010 | |
Vertical Pipetter Puller | David Kopf Instruments | 720 | |
Zebrafish microinjection mold | Adaptive Science Tools | i34 |