Classic genetic studies are based on correlating genetic alterations with the resulting phenotypes. Several important signaling pathways, including mTOR, apoptosis, autophagy and Hippo pathways, have been discovered by classic genetics. Given the fact that some lethality-causing genes or essential genes are impossible to overexpress or knock out, fine-tuning their expression is necessary for the analysis of their function. Moreover, the irreversible manipulation of gene expression often drives compensatory adaptation in higher organisms. Therefore, the ability to switch the gene expression on and off or to modulate the level of gene expression in a quantitative and temporal way can preferentially reveal the direct consequence of a certain genetic change and provide an additional filter to exclude other side- and off-target effects. This is especially beneficial when working with mammalian cells that are maintained and controlled by highly intricate genetic networks.

1. Induction of Target Gene The above-described

lacR/O-based systems were soon found to be too limited due to their inefficiency and moderate potency in mammalian cells. Even though a chimeric lacR-VP16 has been described to activate a minimal promoter almost 1000-fold at elevated temperatures in the presence of IPTG, the temperature dependence and the inherent IPTG-related problems were found to limit the usability of this approach. Soon after, another bacterial regulatory element, the Tn10-specified tetracycline-resistance operon of E. coli, was found to exhibit a superior performance and became a popular tool to control mammalian gene expression. Currently, there are three configurations of this system: (1) The repression-based configuration, in which a Tet operator (TetO) is inserted between the constitutive promoter and gene of interest and where the binding of the tet repressor (TetR) to the operator suppresses downstream gene expression. In this system, the addition of tetracycline results in the disruption of the association between TetR and TetO, thereby triggering TetO-dependent gene expression. (2) Tet-off configuration, where tandem TetO sequences are positioned upstream of the minimal constitutive promoter followed by cDNA of gene of interest. Here, a chimeric protein consisting of TetR and VP16 (tTA), a eukaryotic transactivator derived from herpes simplex virus type 1, is converted into a transcriptional activator, and the expression plasmid is transfected together with the operator plasmid. Thus, culturing cells with tetracycline switches off the exogenous gene expression, while removing tetracycline switches it on (Figure 1B). (3) Tet-on configuration, where the exogenous gene is expressed when tetracycline is added to the growth medium. Even though tetracycline is nontoxic to mammalian cells at the low concentration required to regulate TetO-dependent gene expression, its continuous presence is suboptimal in a variety of experimental setups. Moreover, the regulation is usually slow when the effector has to be removed by multiple washes [23]. Thus, a mutant tTA with four amino acid substitutions, termed rtTA, was developed by random mutagenesis of tTA. Unlike tTA, rtTA binds to TetO sequences in the presence of tetracycline, thereby activating the silent minimal promoter. Based on the three configurations described above, several additional optimizations have been made. One of these was an attempt to further reduce the leakage of the system. In the repression-based configuration, transcriptional repressor domains, such as the Krüppel-associated box (KRAB) of human KOX1, have been fused with tetR to reduce the leakage. In the Tet-on configuration, newly engineered rtTAs with few mutations make them exponentially active and sensitive. More importantly, these rtTA variants show no activity in the absence of doxycycline (a synthetic tetracycline derivative). Recently, another mutation, TetRI194T, on top of the above rtTAs, was shown to have an even more superior performance. However, there is still a major drawback associated with the tetracycline-induced operator system. That is that upon continuous cell culture, some cell lines can spontaneously lose their inducibility, especially after successive selection rounds. Finally, it should also be noted that tetracycline-derived contaminants that are often present in cell culture serums can cause problems with tetracycline-based expression systems. These can, however, be avoided using tetracycline-free fetal bovine serum.

2. Induction of Knockdown or Knockout of Target Gene

Tetracycline-controlled inducible operator systems can also be combined with RNA interference

and CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats-CRISPR associated

protein 9) to knock down and knock out gene expression, respectively, in mammalian cells. RNA

interference (RNAi) has emerged as an essential tool to achieve knockdown of gene expression [31,32].

It employs a small double-strand RNA processed by endoribonuclease DICER to trigger RNA-induced

silencing complex (RISC)-dependent mRNA degradation, thereby leading to the subsequent decline of

corresponding protein [33]. This can be activated by two means: the delivery of synthetic siRNAs,

which induces a transient knockdown of protein expression, or by expressing short hairpin RNA

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(shRNA), which can be processed by RNAi machinery into siRNA in vivo. Stable transfection of shRNA

expressing plasmids into mammalian cells can constitutively knock down specific gene expression [33].

However, in the case where a gene’s knockdown has a deleterious effect on target cells, the inducible

expression of shRNA achieved by repression based configuration becomes a more reliable approach [34].

A minor adjustment has to be made to avoid the leaky expression of shRNA driven by RNA-Pol

III-dependent promoters (H1 or U6) in the absence of tetracycline, which is two tetracycline operons

that need to be placed flanking the TATA box [35,36]. Corresponding lentiviral systems have also been

developed for cells that are difficult to transfect [34].

The CRISPR-Cas9 technology has recently revolutionized gene editing. Cleavage of specific DNA

site catalyzed by Cas9 endonuclease followed by error-prone non-homologous-end-joint repair can

efficiently result in gene knockout [37,38]. Original protocol to generate knockout cells by CRISPR

technology requires the selection of positive and negative clones for phenotypic comparison. More

than two-three weeks of culturing under selection pressure fosters cells adapted to the loss of the

gene of interest. This adaptation may also involve uncontrolled irreversible changes in other genes,

if these are advantageous for the survival of the knockout cells. Moreover, the frequently observed

clonal variation can make it challenging for researchers to draw reliable conclusions by analyzing the

phenotypes of single-cell-derived clones. Thus the inducible expression of Cas9 driven by rtTA can

overcome these drawbacks. Comparing non-induced and induced cells within a short time-frame

tends to reveal the direct effects caused by the loss of the gene of interest.