Stable lines of transgenic zebrafish exhibit reproducible patterns of transgene expression

Abstract

To study the frequency of germ-line transformation and to examine the reproducibility of tissue-specific transgene expression, we produced several lines of transgenic zebrafish expressing a recombinant chloramphenicol acetyltransferase (CAT) gene. Supercoiled plasmids containing both Rous sarcoma virus and SV-40 promoter sequences upstream of the CAT coding region were injected into zebrafish embryos prior to first cleavage. CAT activity could be detected in batches of injected embryos as early as 8 h and up to at least 12 days post-fertilization. Approximately 18% of injected fish raised to maturity exhibited CAT activity in their fins, and approximately 5% of injected fish became stable germ-line transformants. Breeding studies indicated that although transgenic founder fish were frequently germ-line mosaics, transgenic individuals of subsequent generations were fully hemizygous for the transgene marker. The transgenes present in the F1 progeny of four independent lines were relatively well expressed in fin and skin, while lower levels of expression were observed in heart, gill and muscle. Little or no CAT expression was observed in the brain, liver and gonad. A monoclonal antibody directed against the CAT gene product consistently revealed variegated patterns of CAT expression in ectodermally derived fin epidermal cells in three of these lines. These results show that it is possible to efficiently produce stable germ-line transformants of the zebrafish and to observe reproducible tissue-specific patterns of transgene expression in this organism. Possible mechanisms for the variegated expression observed within tissues are also considered.

Endogenous protein tagging in medaka using a simplified CRISPR/Cas9 knock-in approach

“…The eGFP-cbx1b and mGL-cbx1b KI lines serve as endogenous ubiquitous nuclear markers ( Nielsen et al, 2001 ; Lomberk et al, 2006 ). The generation of truly ubiquitous lines by transgene overexpression in teleost fish ( Centanin et al, 2014 ; Burket et al, 2008 ) is a difficult endeavour and requires constant monitoring for variegation and silencing ( Goll et al, 2009 ; Akitake et al, 2011 ; Burket et al, 2008 ; Stuart et al, 1990 ). Yet these ubiquitous fluorescent reporter lines are invaluable tools for researchers.…”

“…First, by using endogenous fusion proteins there is no requirement for overexpression of cell cycle regulators. Second, the potential issue with transgene variegation and silencing is avoided ( Akitake et al, 2011 ; Goll et al, 2009 ; Burket et al, 2008 ; Stuart et al, 1990 ). Finally, utilizing a single-colour cell cycle reporter allows its simultaneous use with other fluorescent reporters during live-imaging experiments.…”

Single-cell transcriptional dynamics in a living vertebrate

“…While transient expression of transgenes via injection of one-cell stage embryos is common in zebrafish (Campbell et al 2015), this approach is not ideal for reproducible, quantitative measurements of transcription due to the possibility of positional effects and other sources of variation from injections (Stuart et al 1990; Roberts et al 2014). Therefore, we generated two stable transgenic zebrafish lines via I-SceI meganuclease transgenesis (Soroldoni, Hogan, and Oates 2009): one carrying a reporter construct containing MS2 stem loop sequences inserted downstream of the her1 regulatory region, dubbed “ her1 -MS2”, and another carrying an MCP-mNeonGreen (herein “MCP”) fusion driven by a ubiquitin promoter (Fig.…”

From multiallele fish to nonstandard environments, how ZFIN assigns phenotypes, human disease models, and gene expression annotations to genes

“…Zebrafish are also amenable to transgene insertion to knock out genes ( Amsterdam et al 2004 ), overexpress endogenous or other species genes ( Sabaawy et al 2006 ; Padanad et al 2012 ), insert mutant genes ( Kimelman et al 2017 ; Endo et al 2022 ), or express fluorescent proteins to mark anatomical structures ( Lawson and Weinstein 2002 ; Clark et al 2011 ). Transgene insertion is accomplished by the injection of DNA constructs (transgenic constructs) into zebrafish embryos, which are then raised to maturity and screened for stable germline transmission ( Stuart et al 1990 ; Culp et al 1991 ). ZFIN creates records for transgenic constructs and makes an association with the transgenic genomic features (alleles) using a phenotypic or innocuous relationship.…”