PUBLICATION
An optimized QF-binary expression system for use in zebrafish
- Authors
- Burgess, J., Burrows, J.T., Sadhak, R., Chiang, S., Weiss, A., D'Amata, C., Molinaro, A.M., Zhu, S., Long, M., Hu, C., Krause, H.M., Pearson, B.J.
- ID
- ZDB-PUB-200724-15
- Date
- 2020
- Source
- Developmental Biology 465(2): 144-156 (Journal)
- Registered Authors
- Burgess, Jason, Burrows, Jeff, Chiang, Sharon, D'Amata, Cass, Hu, Chun, Krause, Henry, Molinaro, Alyssa, Pearson, Bret, Sadhak, Roshan, Weiss, Alex
- Keywords
- Binary expression system, Gal4, Gene tagging, Knock-in, Lineage tracing, Q-system, QF, QUAS, Zebrafish
- MeSH Terms
-
- Animals
- Animals, Genetically Modified*/genetics
- Animals, Genetically Modified*/metabolism
- Gene Expression*
- Neurospora crassa/genetics*
- Protozoan Proteins*/genetics
- Protozoan Proteins*/metabolism
- Transcription Factors*/genetics
- Transcription Factors*/metabolism
- Zebrafish*/genetics
- Zebrafish*/metabolism
- PubMed
- 32697972 Full text @ Dev. Biol.
Citation
Burgess, J., Burrows, J.T., Sadhak, R., Chiang, S., Weiss, A., D'Amata, C., Molinaro, A.M., Zhu, S., Long, M., Hu, C., Krause, H.M., Pearson, B.J. (2020) An optimized QF-binary expression system for use in zebrafish. Developmental Biology. 465(2):144-156.
Abstract
The zebrafish model organism has been of exceptional utility for the study of vertebrate development and disease through the application of tissue-specific labelling and overexpression of genes carrying patient-derived mutations. However, there remains a need for a binary expression system that is both non-toxic and not silenced over animal generations by DNA methylation. The Q binary expression system derived from the fungus Neurospora crassa is ideal, because the consensus binding site for the QF transcription factor lacks CpG dinucleotides, precluding silencing by CpG-meditated methylation. To optimize this system for zebrafish, we systematically tested several variants of the QF transcription factor: QF full length; QF2, which lacks the middle domain; QF2w, which is an attenuated version of QF2; and chimeric QFGal4. We found that full length QF and QF2 were strongly toxic to zebrafish embryos, QF2w was mildly toxic, and QFGal4 was well tolerated, when injected as RNA or expressed ubiquitously from stable transgenes. In addition, QFGal4 robustly activated of a Tg(QUAS:GFPNLS) reporter transgene. To increase the utility of this system, we also modified the QF effector sequence termed QUAS, which consists of five copies of the QF binding site. Specifically, we decreased both the CpG dinucleotide content, as well as decreased the repetitiveness of QUAS, to reduce the risk of transgene silencing via CpG methylation. Moreover, these modifications to QUAS removed leaky QF-independent neural expression that we detected in the original QUAS sequence. To demonstrate the utility of our QF optimizations, we show how the Q-system can be used for lineage tracing using a Cre-dependent Tg(ubi:QFGal4-switch) transgene. We also demonstrate that QFGal4 can be used in transient injections to tag and label endogenous genes by knocking in QFGal4 into sox2 and ubiquitin C genes.
Genes / Markers
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping