The inducible lac operator-repressor system is functional in zebrafish cells

Background Zebrafish are a foundational model organism for studying the spatio-temporal activity of genes and their regulatory sequences. A variety of approaches are currently available for editing genes and modifying gene expression in zebrafish, including RNAi, Cre/lox, and CRISPR-Cas9. However, the lac operator-repressor system, a component of the E. coli lac operon which has been adapted for use in many other species and is a valuable, flexible tool for studying the inducible modulation of gene expression, has not previously been tested in zebrafish. Results Here we demonstrate that the lac operator-repressor system robustly decreases expression of firefly luciferase in cultured zebrafish fibroblast cells. Our work establishes the lac operator-repressor system as a promising tool for the manipulation of gene expression in whole zebrafish. Conclusions Our results lay the groundwork for the development of lac-based reporter assays in zebrafish, and adds to the tools available for investigating dynamic gene expression in embryogenesis. We believe that this work will catalyze the development of new reporter assay systems to investigate uncharacterized regulatory elements and their cell-type specific activities.


Background
Experimental approaches for the study of transcriptional regulation by cis-regulatory elements in vivo require methods for both genetically modifying cells or organisms, and for measuring expression levels of specific genes. Zebrafish (Danio rerio) is an ideal model organism for investigating the spatio-temporal-specific regulation of gene expression throughout the developing embryo as it satisfies the requirements for ease of genetic manipulation and expression readout. Microinjection of DNA into fertilized embryos allows for simple and effective delivery of genome-modification tools, such as Tol2 transposons, that mediate genomic integration of constructed expression cassettes. Additionally, the transparency of zebrafish embryos facilitates the observation of fluorescent signals from reporter genes within live cells and tissue. Due to its benefits as a model organism, many technologies for studying gene function have been developed in zebrafish, including Cre/lox [1], tamoxifen-inducible Cre [2], the Tet-On system [3], RNAi [4,5], and more recently, CRISPR based-methods [6]. However, the use of the lac operator-repressor system, a tool which functions transiently in a native context with minimal disruption of local regulation compared to many of the aforementioned methods, has yet to be demonstrated in zebrafish.
The lac operator-repressor system is an inducible repression system established from studies of the lac operon in Escherichia coli (E. coli) that regulates lactose transport and metabolism [7].
The Lac repressor (LacI) binds specifically to a lac operator sequence (lacO), inhibiting the lac promoter and lac operon expression through steric hindrance [8]. Addition of the allosteric inhibitor Isopropyl β-d-1-thiogalactopyranoside (IPTG) to cells frees the lac operon to express its associated gene by inhibiting the binding of LacI to lacO sequences. The use of IPTG with the lac operator-repressor allows for inducible reversal of transcriptional repression.
Since its discovery in prokaryotes, the lac operator-repressor system has been modified for use in eukaryotic organisms to study the regulation of gene transcription [8][9][10][11]. Experiments in mammalian cell lines from mouse, monkey, and human [8][9][10]12], as well as in whole mouse [13], demonstrate the utility of the lac operator-repressor system. It has also successfully been applied in cell lines and whole organisms of the amphibian axolotl, suggesting that this system can be utilized in a wide range of organisms [14]. Modifications to the lac operator-repressor system has allowed for constitutive, ubiquitous expression [8,15,16], visually assessed output [10,12], and the ability to study both gene repression and activation [17,18], emphasizing its flexibility for studying gene expression dynamics. The ability of IPTG to relieve repression in the lac system makes it a more adaptable tool for studying the temporal dynamics of gene expression, compared to constitutively active or repressed reporter gene systems.
In this paper, we provide evidence that the lac operator-repressor system can function in the zebrafish fibroblast cell line PAC2, adding a versatile new tool for the study of zebrafish genetics and transcriptional regulation. The results in a zebrafish cell line support the potential functionality of the lac operator-repressor system to function in whole zebrafish. In addition, we demonstrate that the CMV-SV40 enhancer-promoter produces strong, widespread reporter gene expression in both a zebrafish cell line and embryos. This enhancer-promoter combination provides a flexible, non-tissue specific expression module for zebrafish to aid in reporter gene detection at a cellular level.

The CMV-SV40 enhancer-promoter shows widespread expression in zebrafish
In order to promote the repression of a reporter gene in our assay, we sought to increase LacI expression in transfected cells by including a strong enhancer-promoter driving LacI. The CMV enhancer and promoter are frequently used in reporter vector construction across a wide range of studies due to their strong and constitutive promotion of gene expression. This includes zebrafish where the CMV enhancer-promoter has previously been shown to have potent tissuespecific expression [19]. However, recent studies have demonstrated that the tissue-specific promoter function of non-CMV promoters may be lost when paired with the CMV enhancer [20,21]. With the goal of identifying enhancer-promoter pairs driving non-tissue specific gene expression in whole zebrafish, we examined CMV enhancer activity paired with a non-CMV promoter, the SV40 minimal promoter. The function of the CMV-SV40 enhancer-promoter was first validated in PAC2 cells by inserting them upstream of luciferase in a pGL3 plasmid. Relative luciferase output of the CMV-enhanced SV40 pGL3 plasmid was compared to a pGL3 plasmid containing only a minimal SV40 promoter. The CMV-SV40 enhancer-promoter was able to drive a 24-fold increase in luciferase expression compared to the promoter-only control, suggesting the CMV-SV40 enhancer-promoter is able to function as a strong enhancer-promoter combination in PAC2 cells (Fig. 1A).
To determine if the CMV-SV40 enhancer-promoter was able to enhance reporter expression in whole zebrafish, the Tol2 transposon system was utilized to integrate eGFP-expressing test plasmids into zebrafish embryos. As in PAC2 cells, two constructs were evaluated; one containing a CMV-SV40 enhancer-promoter upstream of an eGFP reporter gene, and one with only a minimal SV40 promoter. While no detectable level of eGFP activity in the promotor-only control was observed (Fig. 1B), composite brightfield and GFP images of 24, 48 and 72 hours postfertilization embryos injected with the CMV-SV40 enhancer-promoter construct showed nontissue specific eGFP expression (Fig. 1C).

The lac operator-repressor system is functional in the PAC2 zebrafish cell line
To test the functionality of the lac operator-repressor system in zebrafish, a repressible reporter plasmid containing 6 lac operators in the 5'UTR of the firefly luciferase gene and a LacIexpressing plasmid were co-transfected into PAC2 cells. When a plasmid expressing a nonfunctional LacI (NFLacI) gene was co-transfected, no repression was observed (Fig. 2), whereas a plasmid expressing CMV enhancer-driven levels of LacI resulted in about 65% repression. An This indicates that LacI is responsible for repression of luciferase expression in these cells.
To compare PAC2 repression levels to previously published data [22] and test broad functionality within different cell lines, we replicated the LacI experiment in the K562 human cell line. When both cell types were co-transfected with the same plasmid mixture, the performance of the lac operator-repressor system was nearly identical in PAC2 and K562 cells (Sup. Fig. 2). Both PAC2 and K562 cells showed around 60-65% repression when co-transfected with a molar equivalent of CMV enhancer-driven LacI containing plasmid (~400ng), and roughly 10-20% repression when co-transfected with a similar molar equivalent of SV40 promoter-only driven LacI-expressing plasmid. These results demonstrate that the lac operator-repressor system functions in PAC2 cells at a comparable level to human K562 cells.

Discussion
Zebrafish are a commonly used model organism for studying the spatio-temporal dynamics of cisregulatory element activity and gene function. However, the flexible and widely used lac operatorrepressor system has previously been untested in zebrafish. Here we demonstrate that the lac operator-repressor system functions in zebrafish cells, consistent with observed activity in other model eukaryotic systems.
For the development of a reporter system in whole organisms like zebrafish, it is critical to demonstrate non-tissue specific activity of an enhancer to provide robust output for single-cell reporter signal detection. The CMV enhancer is routinely used in reporter assays to drive strong and constitutive gene expression, and in a more widespread nature when paired with a non-CMV promoter [20,21]. We provide quantitative evidence that the CMV-SV40 enhancer-promoter robustly increases luciferase gene expression over an SV40-only control plasmid in zebrafish fibroblasts. Furthermore, strong, non-tissue specific eGFP signal was observed in fertilized zebrafish eggs that persisted 72 hours post-fertilization after integration of a CMV enhancer controlled SV40-eGFP reporter construct. These robust levels of expression are critical in whole-organism studies where only a small number of cells may be expressing a reporter gene, and a high level of expression from a non-tissue specific enhancer-promoter, such as CMV-SV40, may facilitate their detection.
Changes in expression of the repressor protein LacI are inversely related to changes in reporter expression. This response appears to provide a level of repression directly related to the LacI level, rather than functioning as an on/off switch. This will allow for a more nuanced measure of lac regulatory control. Upon the addition of IPTG, luciferase signal was recovered to the level of a non-functional LacI control, indicating that robust repression is completely reversible at low IPTG concentrations. The pronounced response to IPTG treatment, as well as minimal toxicity in a zebrafish cell line, suggest the lac operator-repressor system is a viable tool for use in whole zebrafish.
Lac operator-repressor systems can be used to control endogenous gene expression without interrupting native regulatory processes, as lacO sites can be inserted in benign regions, such as introns and UTRs. Transcriptional inhibition of RNA polymerase by steric hindrance can achieve repression without introducing artificial modifications to the locus and causing prolonged alterations in regulatory behavior. This is in contrast to other systems that achieve transcriptional control by tethering a protein domain with activating or silencing effects through chromatin modifying or other endogenous mechanisms. Specificity of repression is also less of a concern compared to novel CRISPRi methods known for off-target effects [23]. As demonstrated by the REMOTE-control system, the lac system can also be used in conjunction with Tet-related systems to drive both activation and repression of a single loci, bringing additional flexibility to zebrafish studies [17]. This system allows for time-controlled experiments, where a reporter gene is repressed only for a limited time window, making it a crucial tool for replicating the restriction of gene expression during development.

Plasmid design
CMV-SV40 enhancer-promoter luciferase plasmids were generated by restriction digestion to insert a CMV enhancer and a minimal SV40 promoter, or only a minimal SV40 promoter, upstream of a luciferase reporter molecule in the context of a pGL3 plasmid (Promega, E1751). Plasmids Lac operator-repressor system plasmids were created using the EMMA golden gate assembly method [25]. All plasmids assembled using EMMA have a backbone consisting of an ampicillin resistance gene and a high-copy-number ColE1/pMB1/pBR322/pUC origin of replication.
Backbone elements are denoted by terminating dotted lines in all plasmid schematics (Fig. 1, Fig.   2, Sup. Fig. 1, Sup. Fig. 2). The EMMA toolkit was a gift from Yizhi Cai (Addgene kit # 1000000119) [25]. The lacI CDS and C-terminal NLS were cloned from the Addgene plasmid pKG215 and inserted into an EMMA entry vector to create an EMMA part. pKG215 was a gift from Iain Cheeseman (Addgene plasmid # 45110) [26]. A frameshift mutation was introduced by inserting an adenosine in the fourth codon of lacI to create a non-functional LacI (NFLacI) for use in control experiments. The LacI-expressing module contains a minimal SV40 promoter, the lacI gene, and a SV40 polyA tail, with or without the addition of an upstream CMV enhancer (Fig. 2).
The repressible reporter plasmid includes a CMV enhancer and a minimal SV40 promoter upstream of a firefly luciferase gene with symmetric lac operators inserted in its 5'UTR, terminated by a SV40 polyA tail. In order to maximize repression activity, six copies of the lac operators containing the sequence AATTGTGAGCGCTCACAATT were utilized in this study. This sequence is the "symmetric" lac operator that possesses tighter binding with LacI than the canonical lac operator sequences [17].

Cell culture
The zebrafish fibroblast cell line PAC2 was maintained as previously reported [27].

Electroporation and luciferase reporter assay
To assess the activity of the CMV enhancer in zebrafish cell culture, 4000ng of firefly luciferase expressing plasmids either with or without the CMV enhancer were transfected into 2x10 6 PAC2 cells via electroporation (Fig. 1A). 100ng of the renilla luciferase expressing plasmid (pRL-SV40 Promega, E2231) was included as a transfection control. Firefly/renilla luciferase signal was calculated as the mean of ratios of three technical replicates per biological replicate. Fold change was then calculated relative to the signal of the SV40 promoter-only containing plasmid. The mean of fold-changes is reported and error bars represent standard deviation.
To test the functionality of our dual module lac repressor system in zebrafish cell culture, 2000ng repressible module and 2000ng of LacI-expressing plasmid were co-transfected into 2x10 6 PAC2 cells by electroporation. 400ng of pRL-SV40 was included as a transfection control (Fig. 2).

Zebrafish microinjections
Microinjections were carried out using the Tol2 transposon system as previously described [24,28]. Zebrafish embryos were co-injected within 30 minutes of fertilization in the single-cell stage with Tol2 mRNA, the experimental plasmid, and phenol red for visualization. All embryos were maintained in 1X Holt buffer and fluorescent activity assessed at 24, 48, and 72 hours postfertilization.

Declarations Ethics Approval and consent to participate
This research was approved by the University of Michigan's University Committee on Use and Care of Animals (protocol number PRO00008385).

Consent for Publication
Not applicable

Availability of Data and Materials
Data sharing is not applicable to this article as no datasets were generated or analysed during the current study.

Competing Interests
The authors declare that they have no competing interests.

Supplemental Material Supplemental Methods Cell Culture
The human myelogenous leukemia cell line K562 was cultured in RPMI 1640 + glutamine (ThermoFisher, 11875093) supplemented with 10% heat inactivated fetal bovine serum and 1% antibiotic-antimycotic until confluent. During growth K562 were incubated at 37°C in 5% CO2. Cells were split every two days into fresh media to avoid extended confluence.
To compare the LacI dual module between different human and zebrafish cell types, we cotransfected 500ng of repressible module with 500ng of LacI-expressing plasmids into 1x10 6 K562 cells or PAC2 cells by electroporation. 100ng of pRL-SV40 plasmid was included as a transfection control (Sup. Fig. 2).

Supplemental Figures
Supplemental Figure 1. High levels of IPTG negatively impact the output of the lac operatorrepressor system in K562 cells. Co-transfections of repressible reporter plasmids with equimolar amounts of CMV-SV40 enhancer-promoter enhancer driven LacI-expressing plasmids were exposed to increasing levels of IPTG. At 1mM IPTG, the output signal is restored to the level observed when NFLacI-expressing plasmids are co-transfected with repressible reporter plasmids, indicating that 1mM IPTG is sufficient to relieve LacI repression. At IPTG concentrations >1mM an increasingly lower output of signal relative to NFLacI levels was observed. The TSS begins where the SV40 promoter begins to slope downward. Error bars represent standard deviation of replicates (n=3). Points represent values for all 3 replicates in each condition. The dashed line shows the NF LacI IPTG-negative control.
Supplemental Figure 2. The lac operator-repressor system performs similarly in both human and zebrafish cell lines. K562 and PAC2 cells transfected with the same lac operator-repressor plasmid mixtures result in similar repression profiles. The promoter-only LacI-expressing plasmids resulted in roughly 10-20% repression in both cell types and the CMV-SV40 enhancer-promoter driven LacI-expressing plasmids resulted in ~60% repression in both cell types. For each of the 3 plasmid combinations above, 100ng of pRL, 4:1 molar equivalents of repressible module plasmid:pRL, and 4:1 molar equivalents of LacI-expressing plasmid:pRL were co-transfected into 1 million K562 cells or PAC2 cells. 6 biological replicates were performed for each condition and 3 were exposed to 1mM IPTG and the remaining 3 were not exposed to IPTG. The TSS begins where the SV40 promoter begins to slope downward. Error bars represent standard deviation of replicates (n=3). Points represent values for all 3 replicates in each condition. The dashed line shows the NF LacI IPTG-negative control.