The use of genetically modified mice is crucial for the study of gene function both in health and disease, as gene expression is manipulated by achieving overexpression or deletion of the gene in the wild-type or mutated form, thus finding its real function. On the other hand, the endogenous gene locus can be disrupted or changed in slight ways using techniques such as homologous recombination in embryonic stem cells which are then cultured to produce genetically modified mice.
Loss-of-Function Studies
When a phenotype results from the deletion or modification of a given gene, it is part of a loss-of-function study of the gene. The difficulty is that phenotypes are often the result of the overlapping expression of more than one gene from the same family or are altered by other events which are poorly understood, but which allow an organism to develop with functional capabilities. These capabilities contain a random modified gene or transgene which could be harmful (a null allele) or destructive.
This means that constitutive expression of a loss-of-function phenotype may be different from that which occurs when the adult organism suffers the sudden loss of the same gene, either as a whole or in a single tissue. For this reason, researchers would like to have more control over the period and location of gene deletion or overexpression.
Some possible mechanisms include the use of the Cre-loxP system (site-specific DNA recombinase) which helps to integrate, delete, or achieve the inversion of a DNA fragment, whether endogenous or imported, and in a controlled environment.
Another method is the use of CRE-dependent recombination, which allows for conditional allele ablation specific for the cell type and timing, and for the activation of inserted transgenes in controlled fashion. With CRISPR/Cas9 technology, it is even possible to target more than one gene in living cells.
However, there are difficulties with all these techniques, especially the artificially elevated transgene expression levels which may be harmful to some types of cells, and the tendency of CRE recombinase to bind to and cleave genomic DNA in nonspecific manner which may kill the cell.
The Cas9 is an endonuclease and it too may bind multiple gene loci stochastically. All these are reasons why phenotypic expression in GM mice may be different from the function of the same gene in adults or even in the same tissue by itself.
This is the cause for the rise of interest in enzyme systems which permit the researcher to insert or ablate genes of interest in a timed and graded manner, or even reversibly. Inducible transgene RNA interference (RNAi) techniques are promising as a scalable option in genetic modification studies, rather than the traditional transgenic or loss-of-function techniques. Using these techniques, it is possible even to carry out genome-wide RNAi screening in vivo.
Another whole genome technique is the genome-wide RNA-based CRISPR interference (CRISPRi) to interrogate gene function and screen genes. CRISPRi uses a dead enzyme molecule of Cas9 (dCas9) which is fused to a Krüppel-associated box (KRAB) transcriptional repression domain. This complex reduces the expression of the target gene when the dCas9 is bound to the target start site of the gene involved, thus inhibiting transcription.
The biggest obstacle to this approach at present is the design of a functional guide RNA for CRISPRi, for which reason RNAi screening remains the method of choice for reversible gene manipulation.
Tet-On/Off System
In most experimental set-ups, the timed and targeted regulation of transgene and RNAi expression in mice is carried out using the tetracycline-regulated Escherichia coli-derived Tet-On/Off system. Developed by Gossen and Bujard, it is more robust than other comparable systems such as the lac repressor-lac operon-based system.
The system uses a transgene activator or repressor which is tetracycline responsive or doxycycline-responsive, namely, tTA or rtTA, along with a tetracycline-responsive promoter element (TRE) which controls the desired gene of interest.
The first-generation tTA is a hybrid transcription factor derived from the fusion of the Tet repressor TetR from prokaryocytes, which is responsible for DNA binding that can be inhibited or reversed by tetracycline binding (Tet-Off), with the transactivation domain of the transcription factor VP16 from the herpes simplex virus, an acidic domain which drives the expression of the transgene.
However, 1995 saw the launch of another similar system which used a ‘reverse’ transactivator (rtTA) that has four amino acids different from the tTA molecule, located inside the TetR sequence, which reverses the responsiveness to tetracycline or doxycycline and allows DNA binding and induction of transgene expression when tetracycline or doxycycline are added, namely, Tet-On.
A Tet-responsive promoter complex PTET most commonly comprises the CMV minimal promoter with seven operator elements, namely, the tetO from E. coli, which allow the tTA and rtTA27 to bind specifically to DNA in a tetracyclin-responsive manner.
The Tet system is powerful and capable of widespread use, but some limitations have been pointed out which could narrow its application base. For one, the CMV promoter has marked leakiness and is poorly expressed in some types of cells. This is countered by the PTET-Tight from Clontech (the current Takara Bio) which is an alternative promoter system that has seven tetO elements upstream of the CMV minimal promoter sequence from -35 to +12, but without the CMV enhancer sequence.
This makes it remain inactive in transcription unless a tetracycline- or doxycycline-responsive transactivator is present as well. Other changes have been made for the better as well, such as codon optimization and the introduction of a tetracycline-controlled transcriptional silencer (tTS) such as the KRAB domain of the human Kid-1 gene. The tTS, however, is limited to the analysis of cell lines because of its requirement for three separate transgenes which are to be co-expressed.
The greatest utility of the Tet-On/Off system has been in cancer research. It has been used to overexpress oncogenes such as myc, ras or bcl-2 as conditional transgenes, or to silence tumor suppressor genes such as p53, PTEN or adenomatous polyposis coli (APC) using RNAi in a controlled manner.
Genome-wide in vivo RNAi screening has also helped uncover modifiers of oncogenesis, while still other discoveries include lymphocyte development, or the natural history of a lymphocyte using the conditional expression of fluorescent markers such as the green fluorescent protein, GFP.
At the same time, it has some limitations, which include the silencing of transgenes or the induction of compensatory effects that inhibit or reverse RNAi in vivo at protein level, which could negate its usefulness in studying essential genes for those involved in gene maintenance.
All these results are taken together with a report that describes how tetracycline can act as a mitochondrial poison and affect gene expression profiles on a global scale within mammalian cells, to indicate that the Tet system has an inherent system-specific biological bias that can select for clones that are resistant to cell death.
The current experiment confirms this suggestion and indicates the further possibility that Tet-transactivator expression could reduce the survival of antigen-activated T and B cells in mice.
Histology
For this experiment, spleens were fixed in 4% PFA (paraformaldehyde) and then embedded in paraffin. Sections of 3-micron thickness were taken and subjected to hematoxylin-eosin staining for histological examination. Olympus VS120 slide scanning was used to acquire images which were then segmented, and the splenic follicles assessed with the Ilastik object classification tool.
Both GCs and apoptotic cells needed to be visualized at the same time, so paraffin was removed from these sections and rehydrated. The GCs were then stained using fluorescein isothiocyanate (FITC)-conjugated peanut agglutinin following the retrieval of antigens in TE10/1, the pH being 8 at 80 °C for 30 minutes. After this, the sections were soaked in 0.1% Triton X-100/0.1% sodium citrate buffer, before being dehydrated by graded series of alcohol and chloroform.
The apoptotic cells were then detected using TUNEL reaction in a humidified chamber for one hour at 37 °C. The reagents used were recombinant terminal transferase and tetramethylrhodamine (TRITC)-dUTP (both Roche, Manheim, Germany), and the nuclei were counterstained using DAPI (Sigma). Finally, the images were passed through the image analysis software TissueQuest (TQ4.0, TissueGnostics, Vienna, Austria) for quantitative analysis by a blinded observer.
Discussion
The Tet-On/Off system which can induce the conditional expression of protein-coding genes or shRNAs is being used more often in preclinical cancer research models to control transgene expression in a timed and reversible manner.
Other increasingly common applications include exploring the adaptive immune response, such as how the GC reaction is regulated dynamically following infection. It is becoming clear that this system is subject to producing non-specific immune responses which can alter the data interpretation in some experiments, as in this case.
About TissueGnostics
TissueGnostics (TG) is an Austrian company focusing on integrated solutions for high content and/or high throughput scanning and analysis of biomedical, veterinary, natural sciences and technical microscopy samples.
TG was founded by scientists from the Vienna University Hospital (AKH) in 2003. It is now a globally active company with subsidiaries in the EU, the USA and China and customers in 28 countries.
TG systems offer integrated workflows, i.e. scan and analysis, for digital slides or images of tissue sections, Tissue Microarrays (TMA), cell culture monolayers, smears and of other samples on slides and oversized slides, in Microtiter plates, Petri dishes and specialized sample containers.
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