Rapid and Efficient Zebrafish Genotyping Using PCR with High-resolution Melt Analysis

Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic ...

University of Utah School of Medicine

The mouse Pactolus and CD18 genes are highly conserved paralogues. The expression patterns of these genes are diverse in that most cells of hematopoietic lineage express CD18, but Pactolus is only expressed by maturing neutrophils. The minimal promoters of these two genes are homologous, including the conservation of two tandem PU.1-binding sites upstream of the transcriptional start site. To define the means by which these two structurally similar but functionally distinct promoters operate, a series of reporter assays, electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation analyses, were performed. Transfection of Pactolus constructs into mouse macrophages, which do not express Pactolus, defined a negative control element within the first 100 base pairs. The presence of this negative regulatory site, distinct from the PU.1-binding site, was confirmed by EMSA oligonucleotide competition and gene reporter assays of Pactolus/CD18 chimeric constructs. Although PU.1 binding can be detected on Pactolus and CD18 minimal promoter segments with EMSA, only the CD18 promoter shows PU.1 binding in vivo, suggesting that the negative regulatory protein may block PU.1 from binding to the Pactolus promoter, thus inhibiting transcription of the gene. Sequence analysis of the negative control region in the Pactolus promoter suggested potential control by Snail and/or Smad families of transcription regulators. EMSA supershift analysis with antibodies against these proteins, using extracts from macrophages and mucosal mast cells, identified specific binding of Smuc to the promoter element, including a Smuc/PU.1/DNA trimeric complex. These data implicate Smuc as blocking Pactolus transcription in cells expressing PU.1 (and CD18) but not Pactolus.

Transcriptional control of Pactolus: evidence of a negative control region and comparison with its evolutionary paralogue, CD18 (beta2 integrin).

The zebrafish is a powerful experimental system for uncovering gene function in vertebrate organisms. Nevertheless, studies in the zebrafish have been limited by the approaches available for eliminating gene function. Here we present simple and efficient methods for inducing, detecting, and recovering mutations at virtually any locus in the zebrafish. Briefly, double-strand DNA breaks are induced at a locus of interest by synthetic nucleases, called TALENs. Subsequent host repair of the DNA lesions leads to the generation of insertion and deletion mutations at the targeted locus. To detect the induced DNA sequence alterations at targeted loci, genomes are examined using High Resolution Melt Analysis, an efficient and sensitive method for detecting the presence of newly arising sequence polymorphisms. As the DNA binding specificity of a TALEN is determined by a custom designed array of DNA recognition modules, each of which interacts with a single target nucleotide, TALENs with very high target sequence specificities can be easily generated. Using freely accessible reagents and Web-based software, and a very simple cloning strategy, a TALEN that uniquely recognizes a specific pre-determined locus in the zebrafish genome can be generated within days. Here we develop and test the activity of four TALENs directed at different target genes. Using the experimental approach described here, every embryo injected with RNA encoding a TALEN will acquire targeted mutations. Multiple independently arising mutations are produced in each growing embryo, and up to 50% of the host genomes may acquire a targeted mutation. Upon reaching adulthood, approximately 90% of these animals transmit targeted mutations to their progeny. Results presented here indicate the TALENs are highly sequence-specific and produce minimal off-target effects. In all, it takes about two weeks to create a target-specific TALEN and generate growing embryos that harbor an array of germ line mutations at a pre-specified locus.

Simple methods for generating and detecting locus-specific mutations induced with TALENs in the zebrafish genome.

The heteroduplex mobility assay (HMA) is widely used to characterize strain variants of human viruses. To determine whether it can detect small sequence differences in homologous templates, we constructed a series of deletion constructs (1-10 bp deletions) in the multiple cloning site (MCS) of pBluescript II. After PCR amplification of the MCS using a mixture of wild-type and one of the deletion constructs, the resulting PCR amplicons were electrophoresed using 15% polyacrylamide gels. Two types of heteroduplexes exhibited retarded electrophoretic migration compared with individual homoduplexes. Therefore, we applied this HMA to detect transcription activator-like effector nucleases (TALEN)-induced insertion and/or deletion (indel) mutations at an endogenous locus. We found that TALEN in vivo activity was easily estimated by the degree of multiple HMA profiles derived from TALEN-injected F0 embryos. Furthermore, TALEN-injected F0 founder fish produced several unique HMA profiles in F1 embryos. Sequence analysis confirmed that the different HMA profiles contained distinct indel mutations. Thus, HMA is a rapid and sensitive analytical method for the detection of the TALEN-mediated genome modifications.

Efficient identification of TALEN-mediated genome modifications using heteroduplex mobility assays.

MicroRNAs (miRNAs) have quickly emerged as important regulators of mammalian physiology owing to their precise control over the expression of critical protein coding genes. Despite significant progress in our understanding of how miRNAs function in mice, there remains a fundamental need to be able to target and edit miRNA genes in the human genome. Here, we report a novel approach to disrupting human miRNA genes ex vivo using engineered TAL-effector (TALE) proteins to function as nucleases (TALENs) that specifically target and disrupt human miRNA genes. We demonstrate that functional TALEN pairs can be designed to enable disruption of miRNA seed regions, or removal of entire hairpin sequences, and use this approach to successfully target several physiologically relevant human miRNAs including miR-155*, miR-155, miR-146a and miR-125b. This technology will allow for a substantially improved capacity to study the regulation and function of miRNAs in human cells, and could be developed into a strategic means by which miRNAs can be targeted therapeutically during human disease.

Targeting human microRNA genes using engineered Tal-effector nucleases (TALENs).

The Snail family of transcriptional regulators consists of three highly conserved members. These proteins regulate (repress) transcription via the recruitment of histone deacetylases to target gene promoters that possess the appropriate E-box binding sequences. Murine Snai1 is required for mouse development while Snai2 deficient animals survive with some anomalies. Less is known about the third member of the family, Snai3. To investigate the function of Snai3, we generated a conditional knockin mouse. Utilizing Cre-mediated deletion to facilitate the ablation of Snai3 in T cells or the entire animal, we found little to no effect of the loss of Snai3 in the entire animal or in T cell lineages. This finding provided the hypothesis that absence of Snai3 was mitigated, in part, by the presence of Snai2. To test this hypothesis we created Snai2/Snai3 double deficient mice. The developmental consequences of lacking both of these proteins was manifested in stunted growth, a paucity of offspring including a dramatic deficiency of female mice, and impaired immune cell development within the lymphoid lineages.

Deletion of Snai2 and Snai3 results in impaired physical development compounded by lymphocyte deficiency.

Zinc-finger nucleases have proven to be successful as reagents for targeted genome manipulation in Drosophila melanogaster and many other organisms. Their utility has been limited, however, by the significant failure rate of new designs, reflecting the complexity of DNA recognition by zinc fingers. Transcription activator-like effector (TALE) DNA-binding domains depend on a simple, one-module-to-one-base-pair recognition code, and they have been very productively incorporated into nucleases (TALENs) for genome engineering. In this report we describe the design of TALENs for a number of different genes in Drosophila, and we explore several parameters of TALEN design. The rate of success with TALENs was substantially greater than for zinc-finger nucleases , and the frequency of mutagenesis was comparable. Knockout mutations were isolated in several genes in which such alleles were not previously available. TALENs are an effective tool for targeted genome manipulation in Drosophila.

Timothy J. Dahlem

Mutation Generation and Detection Core,
HSC Core Research Facility,
Mutation Generation and Detection Core, HSC Core Research Facility

University of Utah School of Medicine

Rapid and Efficient Zebrafish Genotyping Using PCR with High-resolution Melt Analysis

Timothy J. Dahlem

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University of Utah School of Medicine

Rapid and Efficient Zebrafish Genotyping Using PCR with High-resolution Melt Analysis

Mutation Generation and Detection Core,
HSC Core Research Facility,
Mutation Generation and Detection Core, HSC Core Research Facility