Structured Inquiry-Based Learning: Drosophila GAL4 Enhancer Trap Characterization in an Undergraduate Laboratory Course

Dunne, Christopher R; Cillo, Anthony R.; Glick, D.R.; John, Katherine A; Johnson, Cody L.; Kanwal, Jaspinder; Malik, Brian T.; Mammano, Kristina; Petrovic, Stefan; Pfister, W.; Rascoe, Alexander S.; Schrom, Diane; Shapiro, Scott; Simkins, Jeffrey W.; Strauss, David G.; Talai, Rene; Tomtishen, John Paul; Vargas, Josephine; Veloz, Tony; Vogler, Thomas O.; Clenshaw, Michael E.; Gordon-Hamm, Devin T.; Lee, Kate; Marin, Elizabeth C.

doi:10.1371/journal.pbio.1002030

Cited by 5 publications

(3 citation statements)

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“…The methods of identification of novel Ens are continuously improving, yet the typical En trapping (ET) technology is based on a randomly inserted transposon-mediated vector, which harbors a trapping box containing a mini-promoter, reporter open reading frame (ORF) and polyA, and the trapping box is flanked by inverse terminal repeats (ITRs) of transposon [6,7,8,9,10]. As the development of retrovirus- and transposon-based technologies for gene transfer progresses, the efficiency of integrating reporters into the genome has been improved significantly [11].…”

Section: Introductionmentioning

confidence: 99%

Enhancer Trapping and Annotation in Zebrafish Mediated with Sleeping Beauty, piggyBac and Tol2 Transposons

Shen

Xue

Chan

et al. 2018

Genes

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Although transposon-mediated enhancer trapping (ET) is successfully applied in diverse models, the efficiency of various transposon systems varies significantly, and little information is available regarding efficiency of enhancer trapping by various transposons in zebrafish. Most potential enhancers (Ens) still lack evidence of actual En activity. Here, we compared the differences in ET efficiency between sleeping beauty (SB), piggyBac (PB) and Tol2 transposons. Tol2 represented the highest germline transfer efficiencies at 55.56% (NF0 = 165), followed by SB (38.36%, NF0 = 151) and PB (32.65%, NF0 = 149). ET lines generated by the Tol2 transposon tended to produce offspring with a single expression pattern per line, while PB and SB tended to generate embryos with multiple expression patterns. In our tests, 10 putative Ens (En1–10) were identified by splinkerette PCR and comparative genomic analysis. Combining the GFP expression profiles and mRNA expression patterns revealed that En1 and En2 may be involved in regulation of the expression of dlx1a and dlx2a, while En6 may be involved in regulation of the expression of line TK4 transgene and rps26, and En7 may be involved in the regulation of the expression of wnt1 and wnt10b. Most identified Ens were found to be transcribed in zebrafish embryos, and their regulatory function may involve eRNAs.

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Section: Introductionmentioning

confidence: 99%

Enhancer Trapping and Annotation in Zebrafish Mediated with Sleeping Beauty, piggyBac and Tol2 Transposons

Shen

Xue

Chan

et al. 2018

Genes

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“…The fruit fly has long been an experimental organism of choice not only for introductory undergraduate laboratory classes, but also for upper classes in areas of cell biology , molecular biology , genomics , and biology/chemistry . Recently, the fruit fly was utilized in inquiry and discovery‐based laboratory courses . These courses were developed to combine faculty's ongoing research into laboratory‐based undergraduate curriculum.…”

Section: Discussionmentioning

confidence: 99%

Integration of a faculty's ongoing research into an undergraduate laboratory teaching class in developmental biology

Nam¹

2017

Biochem Molecular Bio Educ

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Traditional developmental biology laboratory classes have utilized a number of different model organisms to allow students to be exposed to diverse biological phenomena in developing organisms. This traditional approach has mainly focused on the diverse morphological and anatomical descriptions of the developing organisms. However, modern developmental biology is focusing more on conserved genetic networks which are responsible for generating conserved body patterns in developing organisms. Therefore, it is necessary to develop a new pedagogical tool to educate undergraduate biology students in the laboratory class of developmental biology with the genetic principles which are responsible for generating and controlling the developing body patterns. A new undergraduate laboratory class for developmental biology was developed in order to offer students the opportunity to explore a wide range of experimental procedures, also incorporating the instructor's on-going research. Thereby the course can serve as a bridge between research and education by combining both into a single theme. The course design involves a sequence of exercises which can be easily adapted to the faculty's ongoing research. This style of laboratory coursework could be a transitional form between a regular laboratory course and a discovery-based laboratory course. © 2017 by The International Union of Biochemistry and Molecular Biology, 46(2):141-150, 2018.

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“…For instance, it has been applied successfully in medaka (Grabher et al, 2003), zebrafish (Balciunas et al, 2004; Scott et al, 2007; Liu et al, 2015), mouse (Choi et al, 2018) and insect (Koelzer, KöLsch & Panfilio, 2014) models. Transposon-driven vectors generally harbor a reporter-protein-encoding region downstream of a minimal promoter, which is flanked by transposon inverted terminal repeats (Bier et al, 1989; Wilson et al, 1989; Dunne et al, 2014). It seems that the choices of minimal promoter and transposon are equally important for ET efficiency.…”

Section: Introductionmentioning

confidence: 99%

Development of enhancer-trapping and -detection vectors mediated by theTol2transposon in zebrafish

Chan¹,

Shen²,

Sang³

et al. 2019

PeerJ

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Enhancers are key transcriptional drivers of gene expression. The identification of enhancers in the genome is central for understanding gene-expression programs. Although transposon-mediated enhancer trapping (ET) is a powerful approach to the identification of enhancers in zebrafish, its efficiency varies considerably. To improve the ET efficiency, we constructed Tol2-mediated ET vectors with a reporter gene (mCherry) expression box driven by four minimal promoters (Gata, Myc, Krt4 and Oct4), respectively. The ET efficiency and expression background were compared among the four promoters by zebrafish embryo injection at the one-cell stage. The results showed that the Gata minimal promoter yielded the lowest basic expression and the second-highest trapping efficiency (44.6% at 12 hpf (hour post-fertilization) and 23.1% at 72 hpf, n = 305 and n = 307). The Krt4 promoter had the highest trapping efficiency (64% at 12 hpf and 67.1% at 72 hpf, n = 302 and n = 301) and the strongest basic expression. To detect enhancer activity, chicken 5′HS4 double insulators were cloned into the two ET vectors with the Gata or Krt4 minimal promoter, flanking the mCherry expression box. The resulting detection vectors were injected into zebrafish embryos. mCherry expression driven by the Gata promoter (about 5%, n = 301) was decreased significantly compared with that observed for embryos injected with the ET vectors (23% at 72 hpf, n = 308). These results suggest that the insulators block the genome-position effects and that this vector is fit for enhancer-activity evaluation. To assess the compatibility between the enhancers and the minimal promoters, four enhancers (CNS1, Z48, Hand2 and Hs769) were cloned upstream of the Gata or Beta-globin minimal promoter in the enhancer-activity-detection vectors. The resulting recombinant vectors were assayed by zebrafish embryo injection. We found that Z48 and CNS1 responded to the Gata minimal promoter, and that Hand2 only responded to the Beta-globin minimal promoter. In contrast, Hs769 did not respond to either the Gata or Beta-globin minimal promoters. These results suggest the existence of compatibility between enhancers and minimal promoters. This study represents a systematic approach to the discovery of optional ET and enhancer-detection vectors. We are eager to provide a superior tool for understanding functional genomics.

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Structured Inquiry-Based Learning: Drosophila GAL4 Enhancer Trap Characterization in an Undergraduate Laboratory Course

Abstract: This education article describes a modular laboratory exercise in which undergraduates use fruit flies to generate novel experimental data while learning to perform advanced molecular techniques.

Cited by 5 publications

References 10 publications

Enhancer Trapping and Annotation in Zebrafish Mediated with Sleeping Beauty, piggyBac and Tol2 Transposons

Enhancer Trapping and Annotation in Zebrafish Mediated with Sleeping Beauty, piggyBac and Tol2 Transposons

Integration of a faculty's ongoing research into an undergraduate laboratory teaching class in developmental biology

Development of enhancer-trapping and -detection vectors mediated by theTol2transposon in zebrafish

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