WHOLISTIC transgenics

Creation of WHOLISTIC transgenic lines

Animals expressing genetically encoded fluorescent sensors enable researchers to image fluctuations in the concentration of intra- or extracellular molecules¹. Many lines have been made targeting neurons, yet few exist with respect to imaging such sensors in the rest of the body. Despite the simplicity of the idea of making a pan-cellular genetically encoded calcium indicator (GECI) line, it was not easily achieved. Ubiquitous promoters had already been identified, and two papers had used these to drive GECI expression^2,³; however, expression was either limited to the few-cell stage or was extremely sparse. We tried three different pancellular promoters (b-actin, ubi, olactb), with them either failing or giving sparse expression, as in part previously reported⁴. To better drive expression, we chose the most promising of the three, the ubiquitin promoter, and added the amplificatory element tTA/TRE (which is less prone to silencing than the usual gal4/uas system, as it is less dense in GC bases). The GCaMP variant GCaMP7f was used⁵. The constructs were injected into fertilized eggs and embryos grown up. Once maturity reached, the animals were bred and screened for germ-line expression. From these adults, the ones whose progeny had the most homogenous pancellular expression profiles were kept. For 1-photon optogenetic experiments, exciting with a shorter wavelength than that used for imaging is preferred in order to minimize unwanted excitation of the opsin; thus, we thus also generated a stable pancellular red GECI line Tg(ubi:tTA;TRE:jREGCO1b) compatible with the use of channelrhodopsin and its variants⁶.

WHOLISTIC Zebrafish transgenics

Pancellular Danio rerio transgenic

Transgenic zebrafish expressing GCaMP7f pancellularly, imaged using spinning-disk confocal microscopy.

WHOLISTIC Danionella transgenics

For WHOLISTIC in adult vertebrates, we engineered a pancellular Danionella cerebrum GCaMP8m line, using a recombinant version of the ubi promoter⁷, Tg(ubb$^R$:jGCaMP8m), which shows even better expression profiles and equally good survival characteristics.

Pancellular Danionella cerebrum transgenic

Transgenic Danionella cerebrum expressing GCaMP8m pancellularly, imaged with spinning-disk confocal microscopy.

Sensors

Calcium sensors have varying properties such as affinity which alters the dynamic range of calcium they are sensitive to. We are now in the process of doing a more careful characterization of these in relation to their use for WHOLISTIC imaging.

References

1. Nihms489004-1, Nature, 2013, Chen, Tsai-wen and Wardill, Trevor J and Sun, Yi and Pulver, Stefan R and Renninger, Sabine L and Baohan, Amy and Schreiter, Eric R and Kerr, Rex A and Orger, Michael B and Jayaraman, Vivek and Looger, Loren L and Svoboda, Karel and Kim, Douglas S
2. Generation of gcamp6s‐expressing zebrafish to monitor spatiotemporal dynamics of calcium signaling elicited by heat stress, International Journal of Molecular Sciences, 2021, Li, Fengyang and Long, Yong and Xie, Juhong and Ren, Jing and Zhou, Tong and Song, Guili and Li, Qing and Cui, Zongbin
3. Imaging early embryonic calcium activity with GCaMP6s transgenic zebrafish, Developmental biology, 2017, Chen, Jiakun and Xia, Li and Bruchas, Michael R. and Solnica-Krezel, Lilianna
4. Colored medaka and zebrafish: Transgenics with ubiquitous and strong transgene expression driven by the medaka $\beta$-actin promoter, Development Growth and Differentiation, 2012, Yoshinari, Nozomi and Ando, Kazunori and Kudo, Akira and Kinoshita, Masato and Kawakami, Atsushi
5. High-performance calcium sensors for imaging activity in neuronal populations and microcompartments, Nature Methods, 2019, Dana, Hod and Sun, Yi and Mohar, Boaz and Hulse, Brad K. and Kerlin, Aaron M. and Hasseman, Jeremy P. and Tsegaye, Getahun and Tsang, Arthur and Wong, Allan and Patel, Ronak and Macklin, John J. and Chen, Yang and Konnerth, Arthur and Jayaraman, Vivek and Looger, Loren L. and Schreiter, Eric R. and Svoboda, Karel and Kim, Douglas S.
6. Sensitive red protein calcium indicators for imaging neural activity, eLife, 2016, Dana, Hod and Mohar, Boaz and Sun, Yi and Narayan, Sujatha and Gordus, Andrew and Hasseman, Jeremy P. and Tsegaye, Getahun and Holt, Graham T. and Hu, Amy and Walpita, Deepika and Patel, Ronak and Macklin, John J. and Bargmann, Cornelia I. and Ahrens, Misha B. and Schreiter, Eric R. and Jayaraman, Vivek and Looger, Loren L. and Svoboda, Karel and Kim, Douglas S.
7. Highly efficient tamoxifen-inducible {Cre} recombination in embryonic, larval and adult zebrafish, , 2024, Bakūnaitė, Edita and Gečaitė, Emilija and Lazutka, Justas and Balciunas, Darius