Position effects won’t blow this pIGLET’s house down!

Summary by Will Anderson: Lalonde, R. L., H. H. Wells, C. L. Kemmler, S. Nieuwenhuize, R. Lerma, A. Burger, and C. Mosimann (2023). "pIGLET: Safe harbor landing sites for reproducible and efficient transgenesis in zebrafish." bioRxiv. doi: https://doi.org/10.1101/2023.12.08.570868

Image credit: Midjourney

The ability to easily generate transgenic model organisms can greatly streamline biological research. While scientists routinely introduce engineered transgenes into the zebrafish genome via the Tol2 system [1,2], this process relies on random integration. Due to this random nature, the transgene’s expression is beholden to the genomic “position effects” of wherever it happens to be incorporated – for instance, a transgene might land in a region prone to being silenced [3], greatly reducing its usefulness. The unpredictability of position effects resulting from random integration makes the creation of transgenic zebrafish lines more labor intensive and less replicable.

In contrast to the Tol2 system, the optimization of the phiC31 integrase system in Drosophila [4] and mouse [5] research has allowed for the targeted integration of transgenes into well-characterized, genomic “safe harbor sites” [6] that exhibit consistent and expression-friendly position effects. phiC31 integrase functions by directionally recombining specific sequences termed “attB” and “attP”. Introduction of the integrase and a transgene vector containing an attB sequence into an embryo harboring the attP landing site at a particular locus leads to site-specific introduction of the transgene. This process not only allows for more precise and predictable transgene expression, but also reduces the amount of labor and number of animals required for generating stable transgenic lines.

Here, Lalonde et al. replaced well-characterized, Tol2-generated transgenes free of problematic position effects with phiC31-compatible landing sites in two different zebrafish lines, terming the sites and new lines phiC31 Integrase Genomic Loci Engineered for Transgenesis (pIGLET) [6]. To accomplish this, the authors used CRISPR-Cas9 to remove each transgene from single-cell F0 embryos, leaving behind remnant Tol2 sequences. Next, by targeting these Tol2 arms with CRISPR-Cas9 in single-cell F1 embryos, they knocked in an attP sequence at the former site of each transgene, creating pIGLET fish. When pIGLET embryos harboring these safe harbor attP sequences are injected with mRNA encoding phiC31 integrase and a vector containing both an attB sequence and a transgene of interest, the integrase will incorporate the transgene into the landing site.

To validate the function of transgenes introduced to pIGLET fish, the authors compared the dynamics of transgene expression in a variety of pIGLET-generated lines to Tol2-generated counterparts with the same transgene. They found that pIGLET-introduced fluorophores, Cre recombinase, and loxP-based transgenes all displayed expression consistent with the profiles of their tissue-specific promoters. The authors also validated enhancer testing in pIGLET fish by generating a line harboring a human enhancer with a mutation associated with congenital heart defects, demonstrating that this mutation leads to a loss of expression of the gene driven by the enhancer. 

In generating pIGLET lines, the authors seek to improve the ease and precision of transgenesis in zebrafish, and hope that their methodology will aid in the development of more safe harbor sites. By facilitating faithful and predictable expression of transgenes, these methods can streamline the exploration of development, cell fate decisions, and a host of other areas of research. 

References

1. Kikuta, H., Kawakami K (2009). “Transient and stable transgenesis using tol2 transposon vectors”. Methods in Molecular Biology 546: 69-84.

2. Kwan, K. M., E. Fujimoto, C. Grabher, B. D. Mangum, M. E. Hardy, D. S. Campbell, J. M. Parant, H. J. Yost, J. P. Kanki and C. B. Chien (2007). "The Tol2kit: a multisite gateway-based construction kit for Tol2 transposon transgenesis constructs." Developmental Dynamics 236(11): 3088-3099.

3. Cabrera, A., H. I. Edelstein, F. Glykofrydis, K. S. Love, S. Palacios, J. Tycko, M. Zhang, S. Lensch, C. E. Shields, M. Livingston, R. Weiss, H. Zhao, K. A. Haynes, L. Morsut, Y. Y. Chen, A. S. Khalil, W. W. Wong, J. J. Collins, S. J. Rosser, K. Polizzi, M. B. Elowitz, M. Fussenegger, I. B. Hilton, J. N. Leonard, L. Bintu, K. E. Galloway and T. L. Deans (2022). "The sound of silence: Transgene silencing in mammalian cell engineering." Cell Systems 13(12): 950-973.

4. Knapp, J. M., P. Chung and J. H. Simpson (2015). "Generating customized transgene landing sites and multi-transgene arrays in Drosophila using phiC31 integrase." Genetics 199(4): 919-934.

5. Tasic, B., S. Hippenmeyer, C. Wang, M. Gamboa, H. Zong, Y. Chen-Tsai and L. Luo (2011). "Site-specific integrase-mediated transgenesis in mice via pronuclear injection." PNAS 108(19): 7902-7907.

6. Lalonde, R. L., H. H. Wells, C. L. Kemmler, S. Nieuwenhuize, R. Lerma, A. Burger and C. Mosimann (2023). "pIGLET: Safe harbor landing sites for reproducible and efficient transgenesis in zebrafish." bioRxiv.