Overview of the reporter genes and reporter mouse models

Abstract

Reporter genes are widely applied in biotechnology and biomedical research owning to their easy observation and lack of toxicity. Taking advantage of the reporter genes in conjunction with imaging technologies, a large number of reporter mouse models have been generated. Reporter mouse models provide systems that enable the studies of live cell imaging, cell lineage tracing, immunological research and cancers etc. in vivo. In this review, we describe the types of different reporter genes and reporter mouse models including, random reporter strains, Cre reporter strains and ROSA26 reporter strains. Collectively, these reporter mouse models have broadened scientific inquires and provided potential strategies for generation of novel reporter animal models with enhanced capabilities.

1. INTRODUCTION

Reporter genes refer to certain genes that encode proteins that can be easily distinguished from a background of endogenous proteins.1 Generally, reporter genes are chosen based on the sensitivity, dynamic range, convenience, and reliability of their assay.2, 3, 4 Reporter proteins can be classified into two categories: nonfluorescent proteins and fluorescent proteins (ie GFP [green fluorescent proteins], RFP [red fluorescent proteins]). Employing the reporter genes, a large number of reporter animal models have also been generated and used in a wide range of research studies. In general, two experimental strategies have been adopted to introduce exogenous genes into animal genomes. Animal genomes can be altered either by random transgenesis or by targeted transgenesis, which relies on direct gene targeting or use of gene editing tools (ie TALENs, CRISPR/Cas9).5, 6, 7 In addition, the conditional reporter animal strains were also developed. In particular, the Cre/loxp system is one of the most commonly use system for generation of conditional reporter animal strains. In the conditional Cre/loxp reporter system, the first reporter gene is flanked by two loxp sites facing the same direction, followed by the second reporter gene.8 In this system, the first reporter gene can be expressed before Cre‐mediated excision, while the second reporter gene can only be expressed after Cre‐mediated excision. Here, we summarized the two categories of reporter genes, mouse random reporter strains, mouse Cre reporter strains and ROSA26 reporter strains.

2. REPORTER GENES

2.1. Non‐fluorescent reporter genes

Chloramphenicol acetyltransferase (CAT) and lacZ gene are commonly employed as nonfluorescent reporter genes. CAT is a bacterial enzyme and the first reporter gene which was used to monitor transcriptional activity in cells.3 Chloramphenicol, an inhibitor of prokaryotic protein synthesis, can be detoxified by CAT through catalyzing the transfer of acetyl groups from acetyl CoA to the 3ʹ‐hydroxyl‐position of chloramphenicol. The advantage of CAT is its stability and lack of endogeneous expression in mammalian cells.9, 10 An automated ELISA can facilitate CAT application; however, the sensitivity of this assay is still not as high as for other reporters.3, 11 The lacZ gene, which encodes a well‐characterized bacterial β‐galactosidase has been the most commonly used reporter gene in molecular biology studies.12 β‐Galactosidase catalyzes the hydrolysis of X‐Gal converting it to a blue product, which can be easily visualized. Therefore, it has the advantage over CAT because the assays tend to be simple.

2.2. Fluorescent reporter genes

Fluorescent reporter genes are used as a tool for biological imaging. The frequently‐used fluorescent reporter genes are green fluorescent protein (GFP) and red fluorescent protein (RFP). The GFP from the jellyfish, Aequorea victoria, discovered in 1962 by Shimomura,13 is a protein composed of 238 amino acid residues (26.9 kDa) that exhibits bright green fluorescence when exposed to light in the blue to ultraviolet range.14, 15 Its discovery triggered intense research interest in the structure, biochemistry, and biophysics of GFP‐like fluorescent proteins, which resulted in an avalanche of scientific reports about fluorescent proteins and their applications to solve a series of basic issues in molecular and cell biology.16 GFP and its variants, such as enhanced yellow (EYFP) and enhanced cyan (ECFP), have been developed and are nowadays used in a wide range of areas.4, 5 GFP has become well established as a marker of gene expression in cell and molecular biology.17 In 1997, Okabe et al.18 generated the first “green mouse,”which expressed enhanced green fluorescent protein (EGFP) driven by a CAG promoter (chicken beta‐actin promoter combined with the cytomegalovirus enhancer element). The successful generation of such ‘green mice’ suggested that EGFP expression is nontoxic in mouse. Variants of green fluorescent protein (EYFP and ECFP) were also rapidly used in mice for living imaging.19, 20 Later GFP and its variants were also applied in other species such as pig.21, 22

The emission spectra of GFP variants (YFP and CFP) are very close and it is difficult to visually differentiate between them with readily available imaging systems.23 In addition, a double reporter system is often required to establish reporter strains; therefore, easily identifiable, spectrally distinct colors, such as red, had to be developed. Over the past few years, a number of RFPs that emit orange, red and far‐red fluorescence have been discovered from anthozoans (corals), and are available for a wide range of biological applications.16, 24, 25 The first RFP isolated from Discocoma sp. was DsRed1.26 Hadjantonakis et al27 tried to generate a DsRed1 transgenic mouse but failed to establish this line, which indicated that DsRed1 was not developmentally neutral or that constitutive transgene expression may not be sustained. Because DsRed1 has slow maturation times and poor solubility, improvements were made for DsRed1 to generate the mutant DsRed S197Y.28 DsRed S197Y is brighter and essentially free from the secondary fluorescence peak, which makes it an ideal reporter for double labeling with GFP. A further improved DsRed variant, DsRed.T3, was produced through random mutagenesis.29 Vintersten et al30 generated an Z/RED ES cell line and the corresponding transgenic reporter mouse, which expresses β‐geo before Cre recombination and DsRed.T3 after Cre excision. These transgenic reporter mice developed normally and DsRed.T3 expression was inherited by their offspring at expected Mendelian ratios. As DsRed.T3 can form multimers, a series of monomeric RFPs were generated subsequently. Campbell et al31 generated the first actual monomeric RFP, monomeric RFP 1 (mRFP1), which was later used for examining the expression of native mRFP1 in ES cells and its germline transmission.32 They found that mRFP1 expression in a wide range of tissues is compatible with normal development and fertility in mRFP1 transgenic mice. Now, many monomeric RFPs improved from DsRed or other fluorescent proteins are available and are also widely applied in biology16 and transgenic reporter strains. Two examples are monomeric cherry (mCherry) and tandem dimer Tomato (tdTomato). mCherry, which is brighter, matures faster, and has higher photostability than mRFP1, has been already used to generate ubiquitous mCherry transgenic reporter lines.33, 34, 35, 36, 37 tdTomato exhibits a short maturation time, greater brightness and folds equivalenty to a monomer, which may minimize toxicity when used in transgenic reporter strains.38 Latterly, Auldridge et al39 reported a versatile novel yellow fluorescent protein (LucY), which may also be used in transgenic reporter mouse models generation.

3. RANDOM REPORTER STRAINS

A series of reporter mice have been generated by random transgenesis. Exogenous DNA with a promoter‐cDNA cassette is either introduced into mouse ES cells via transfection or micro‐injected directly into zygotes.27, 40 Choosing an appropriate promoter is one of the crucial factors for the successful random transgenesis. The most commonly used promoter for ubiquitous expression of a transgene is the CAG promoter.41 However, some studies showed that the CAG promoter might cause non‐ubiquitous or sometimes even silencing effects on expression of transgenes.42, 43 Other promoters, such as the human ubiquitin C (UBC) promoter34, 44 and the ROSA26 promoter,45 are also used for inducing widespread expression of transgenes. Since both UBC and the ROSA26 promoter are derived from endogenous genes, their expression efficiency is lower than the CAG promoter.46 Nevertheless, the recent reports demonstrate that the UBC promoter and ROSA26 promoters with genomic insulators show a more ubiquitous expression of the transgene than the CAG promoter.43, 47

4. MOUSE Cre REPORTER STRAINS

Widely applied in experimental genetics, the Cre/loxP system used alone or in combination with transgenesis technologies allowed generation of conditional genome alterations that are spatially and temporally restricted or activated.48, 49 For example, a double reporter system was developed based on the Cre/loxP system. In the double reporter system, a first reporter gene flanked by two loxP sites, facing the same direction, can be expressed prior to Cre recombination, while the second reporter gene can only be expressed after the Cre recombination takes place. Based on Cre/loxP system, He et al50 further reported a novel dual recombinases system. Regarding this system, the Dre‐rox system allows rigorous control of Cre/loxP recombination, thus enhancing the precision of lineage tracing mediated by Cre/loxP system. By using the Cre/loxP system, a series of double reporter mice expressing a combination of reporter genes including CAG‐CAT‐Z (chloramphenicol acetyltransferase/lacZ),51 Z/EG (lacZ/EGFP),52 Z/AP (lacZ/human alkaline phosphatase)53 have been generated. These Cre random reporter mice are capable of monitoring Cre activity in diverse tissues and cell types. However, when used in the random integration method, those reporter strains showed some drawbacks. Firstly, the expression patterns of reporter lines vary due to different copy numbers and positional effects of the integration sites.54 Moreover, the inserted gene can also be subject to gene‐silencing effects in later offspring.55 Secondly, it is not easy to choose a suitable reporter mouse line owning to differences between laboratories in settings and reporter mice assessment standards.56 Furthermore, reporter mice that show high expression of the fluorescent reporter are often infertile or not viable.57 In order to overcome these issues, the ubiquitously expressed ROSA26 locus was used to generate genetically modified reporter strains.58

5. ROSA26 REPORTER ANIMAL MODELS

5.1. ROSA26 locus

Friedrich et al59 introduced several promoter trap constructs containing fusion lacZ‐neo gene (β‐geo) into mouse ES cells by electroporation or retroviral infection. Embryos from the gene‐trap line ROSAβ‐geo26 (reverse orientation splice acceptor β‐geo 26) showed ubiquitous β‐galactosidase (β‐gal) expression during embryonic development. Zambrowicz et al58 later reported that the gene‐trap vector was integrated into a mouse gene and this gene was subsequently named ROSA26. In mouse, the Rosa26 gene is located on chromosome 6 between THUMPD3 and SETD5 genes and has 3 noncoding transcripts (NR_027008.1, NR_027009.1 and NR_027010.1). ROSA26 transcripts 1 and 2 both contain 2 exons and 1 intron, while transcripts 3 is tail‐to‐tail overlapping (3ʹ to 3ʹ) with the THUMPD3 gene exon 3 (Figure 1). The mouse ROSA26 locus shows ubiquitous transcriptional activity but loss of this gene is not lethal.58 The ubiquitous transcriptional activity of this locus indicates that the genomic region is not affected by chromatin configurations which may cause transcriptional repression of exogenous transgenes. Therefore, this locus is widely used as a permissive site for targeted placement of transgenes in mice,60, 61 with no effect on animal viability or fertility. In mice, transgenes have been introduced into the XbaI site in the first intron of the ROSA26 forward transcript where the presence of a splice acceptor allows the transgene expression to be driven by the ubiquitously expressed endogenous promoter.46 Irion62 and Kobayashi63 demonstrated ubiquitous expression of red‐fluorescent protein cDNA, integrated into the human and rat homolog of the mouse ROSA26 locus through homologous recombination. This indicates that the human and rat ROSA26 locus conserve properties of its orthologs in mouse.

Figure 1.

Mouse Rosa26 genomic locus and its adjacent genes (Thumpd3 and Setd5) on chromosome 6. The red arrowheads indicate orientation of transcription of Rosa26, Thumpd3 and Setd5 and genes are shown with exons and introns. Mouse Rosa26 has 3 transcripts (Accession number: NR_027008.1, NR_027009.1 and NR_027010.1) and the transcript NR_027010.1 contains 3 exons and 2 introns. The 3rd exon of NR_027010.1 is tail‐to‐tail overlapping (3ʹ to 3ʹ) with Thumpd3 gene

5.2. ROSA26 reporter strains

Through homologous recombination in ES cells, a series of reporter genes have been inserted into the Rosa26 locus to generate reporter mouse lines with precisely designed genome modifications (Figure 2). Soriano60 constructed the Rosa26 targeting vector which comprises a splice acceptor sequence (SA), a PGK promoter, a neo expression cassette flanked by two loxP sites with the same direction, followed by a triple polyadenylation sequence to prevent neo cassette transcriptional read‐through, a lacZ gene and a polyadenylation sequence. This Rosa26 reporter construct was then linearized and inserted into a unique XbaI site at approximately 300 bp 5ʹ‐upstream of the original gene‐trap integration site in intron 1 of the mouse Rosa26 locus.60 Thus, a reporter mouse line for monitoring Cre recombinase activity at the Rosa26 locus at desired time points was successfully established. However, the endogenous ROSA26 promoter is weaker than exogenous artificial promoters such as CAG promoter,46, 61 resulting in hardly detectable reporter signals in tissues and cells. Therefore, the CAG promoter is often used in knock‐in reporter lines in order to enhance expression activity at the ROSA26 locus (Figure 1).64, 65, 66 The CAG promoter was shown to yield approximately 8‐ to 10‐fold higher expression levels compared to the endogenous ROSA26 promoter.46 A series of reporter genes driven by the CAG promoter were targeted into the mouse Rosa26 locus to generate ROSA26 reporter lines, such as a multifunctional teal‐fluorescent Rosa26 reporter mouse line,67 which strongly expresses mTFP1 (bright teal fluorescent protein) after Cre and Flp mediated recombination. Another example includes, a global double‐fluorescent Cre reporter mouse,38 which expresses membrane‐targeted tandem dimer Tomato (mT) before Cre‐mediated excision or membrane‐targeted green fluorescent protein (mG) after Cre recombination. All of those ROSA26 reporter strains can be used in live cell imaging, lineage tracing, monitoring Cre activity, and analysis of cell morphology and so on.

Figure 2.

Strategies of targeting reporter genes into the ROSA26 locus. From top to bottom: the wild type ROSA26 locus with the indicated targeting site; the structure of the targeted R26R allele before and after Cre excision of the loxP flanked selection marker with stop cassete; the structure of the targeted R26R‐CAG allele before and after Cre excision of the loxP flanked selection marker with stop cassete, where the CAG promoter is inserted in front of the loxP‐flanked selection marker with stop cassette; the structure of R26R‐SA‐CAG allele before and after Cre excision of the loxP flanked cDNA with stop cassette. loxP sequences are indicated by arrowheads and the ROSA26 exon 1 is shown as black rectangles

In addition, combining the ROSA26 locus with Cre/loxP system, Aya et al68 generated multi‐color fluorescent reporter mice which can be applied for lineage tracing. In these multi‐color fluorescent reporter mice, 4 fluorescent reporter genes (GFP, YFP, RFP and CFP) can be expressed in a random manner after Cre‐mediated DNA excisions and inversions. Szyska et al69 generated dual‐luciferase reporter mouse model expressing an NFAT‐dependent click‐beetle luciferase and a renilla luciferase. This reporter mouse model supports longitudinal and functional monitoring of T cells in vivo. Park et al70 reported estrogen receptor alpha‐iCre mouse line which express codon‐improved Cre (iCre) driven by the Esr1 promoter. They further crossed ROSA26‐LacZ reporter mouse strain with Esr1‐iCre mouse line to characterize the function of lineage‐tracing Esr1‐expressing cells. Plummer et al71 described a new ROSA26 mouse strain for cell ablation by DTA (diphtheria toxin subunit A) which can be switched on by Cre‐dependent flip‐excision (FLEx). Boutet et al72 targeted the Wilms’ tumor gene on the X chromosome (WTX) fused to GFP into the mouse ROSA26 locus and generated a novel ROSA26 mouse strain. This ROSA26 mouse model can conditionally express the WTX in different tissues by crossing with several Cre transgenic mice. Dong et al73 targeted exogenous genes into the ROSA26 locus and generated a ratiometric tdTomato‐GCaMP6f reporter mouse which can be applied in visualizing T‐cell calcium dynamics.

The applications of reporter mouse models are various. For example, reporter animal lines labeled with fluorescent proteins fused to different subcellular localization signals allow for the observation of real‐time states of cells and molecules in specific organelles of living organisms. Lineage tracing is now widely used in stem cell research since it provides information about the cell behavior in the context of intact tissue or organ. It is also a powerful method for understanding tissue development, signals regulating cell‐fate decisions and diseases. The immune system plays a vital role in organisms and has the capacity to recognize and destroy malignant cells and pathogens.74 Reporter animal lines, such as cytokine reporter strains, and immune cell population‐labeled reporter strains are quickly emerging in this field to facilitate immunological studies. In the immune system, cytokines are soluble messenger molecules having important regulatory function.75 For example, IL‐4, which is the hallmark cytokine for Th2 cells, plays an important role in immunity against extracellular pathogens.76 Cytokine reporter strains have been established by placing reporter genes under the control of elements from cytokine genes, thus enabling easy identification of their cellular sources. Based on progress in the development of reporter genes and existing reporter animal models, we believe that abundant novel animal models will be generated in the near future and applied to diverse research fields.

CONFLICT OF INTEREST

None.

AUTHOR CONTRIBUTIONS

All listed authors meet the requirements for authorship. SL conceived and wrote the manuscript. XHZ corrected the manuscript. All authors read and approved the final manuscript.

Li S, Chen LX, Peng XH, et al. Overview of the reporter genes and reporter mouse models. Anim Models Exp Med. 2018;1:29–35. 10.1002/ame2.12008