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The following points highlight the two types of mutation strategies. The Strategies are: 1. Cell Type-Specific Mutation Strategies 2. Inducible Mutation Strategies.
1. Cell Type-Specific Mutation Strategies:
When a null mutation of a gene results in a mutant phenotype, limitations in the interpretation of that phenotype can arise because the gene is inactivated in every cell in which it was expressed in the mouse in the wild-type (WT) state.
Therefore, the observed abnormal phenotype may arise from the absence of the functioning gene product in a peripheral organ system, the peripheral nervous system, or the CNS-i.e., in any of those regions in which the gene is normally expressed. It is possible that the absence of a gene product in the periphery may lead to embryonic lethality, precluding use of the mutant for the studies of neural function.
For genes that are widely expressed within the CNS, it may be difficult to identify neural circuits through which mutations produce behavioural perturbations. The ability to inactivate genes in restricted subpopulations of the cells that normally express them will be a valuable asset in studies to uncover the neural mechanisms underlying neural phenotypes.
Recent efforts have focused on a mutational strategy developed to exert spatial control over the pattern of expression of genetic changes introduced into mice. This approach utilizes somatic cell recombination rather than germ cell (or embryonic stem cell) recombination to inactivate a gene in restricted populations of cells or tissues.
In this approach, a tissue- specific promoter is used to direct expression of one of the site-specific recombines to limit gene inactivation to only those cells expressing the recombinase.
The two recombinase systems that have been utilised for genetic manipulation in mice have been the Flp-frt system from yeast, and the Cre-lox system from bacteriophage P1, with the large majority of reports using this technique utilizing the Cre-lox system.
Cre (cyclisation of recombination) recombinase is a 38- kd protein from bacteriophage P1, which recognises and catalyses reciprocal DNA recombination between two loxP (locus of crossing over of PI) sites. The loxP site is the 34- base pair (bp) recognition sequence for Cre composed of a palindromic 13-bp sequence separated by a unique 8-bp core sequence.
A gene or gene segment with flanking loxP (“floxed”) sites will be excised by homologous recombination in the presence of Cre, leaving a single loxP site marking the point of excision and re-ligation of upstream and downstream DNA.
This approach, then, involves generating two independent lines of mice-a line bearing loxP sites, and a transgenic line in which Cre expression is driven by a tissue-specific promoter. Animals with a gene or gene region of interest flanked by loxP sites (floxed) are generated by gene targeting.
Because the loxP sites are relatively small and placed in intronic regions, they do not typically interfere with normal gene transcription. Of course, WT patterns and levels of expression need to be documented in these floxed mouse lines, because inadvertent placement of lox sites into promoter elements or RNA splice sites could disrupt gene expression.
The Cre mice are most commonly generated by creating a transgenic line of mice in which Cre expression is driven by a tissue-specific promoter.
Variability in transgenic expression patterns requires several lines of Cre mice that need to be generated and assayed for patterns of Cre expression. An alternative strategy is to use gene targeting procedures to place Cre under the control of an endogenous promoter.
The advantage of this approach is that Cre expression should closely approximate the WT expression pattern of the gene it is replacing because the original gene’s promoter remains in its endogenous location.
A potential disadvantage is that Cre may disrupt expression of the gene into which it has integrated. Once a line exhibiting the desired pattern of Cre expression is identified, it is crossed with an appropriate floxed line to commence a breeding strategy resulting in the generation of animals with a restricted pattern of gene inactivation.
Several lines of Cre mice have been reported in which expression is restricted to subpopulations of cells within the CNS. The first example of this approach was the inactivation of the glutamate receptor subunit NMDAR1 in CA1 pyramidal neurons of the hippocampus, with expression in other brain areas mostly intact.
NMDAR1 is the predominant N-methyl-D-aspartate (NMDA) receptor subunit and is widely expressed in most CNS neurons. It had been previously demonstrated that widespread gene inactivation in NMDAR1 null mutants resulted in perinatal lethality.
When the mutation was restricted to hippocampal CA1 neurons, animals were viable and exhibited impaired spatial learning and impaired plasticity at CA1 synapses. Thus, spatial restriction of neural mutations can be used to uncover particular brain regions or cell type through which gene inactivation alters behaviour.
The utility of this approach for producing cell type — specific inactivation of genes is enhanced by the fact that the components of the system produced in one laboratory can be “mixed and matched” with components from another laboratory.
That is, Cre lines generated for use with a particular floxed gene may also be used with other floxed genes when a similar pattern of gene inactivation is desired. Collaborative efforts to generate databases of Cre and floxed lines will speed and simplify the production of animals with restricted patterns of gene inactivation.
2. Inducible Mutation Strategies:
The absence of a gene product throughout development complicates the interpretation of mutant phenotypes. Efforts are currently under way to overcome this limitation through the use of gene expression systems that may be induced in the adult animal.
Strategies are under exploration for achieving this goal using a variety of compounds, such as tetracycline, steroid receptor antagonists, and ecdysone to induce gene expression. Although these approaches have yet to be optimized for general use, this development is likely to be close at hand. The tetracycline system has been the most utilised and best developed approach to inducible gene regulation.
Since the introduction of the Tet system by the Bujard laboratory in 1992, many laboratories have validated the utility of this approach to inducible gene regulation, and many refinements/ improvements in the system have been introduced.
This system is based on the regulatory elements of a tetracycline resistance operon of Escherichia coli, in which the transcription of tetracycline resistance genes is negatively regulated by the tetracycline repressor (tetR).
When tetracycline is present, tetR binds the tetracycline and loses its capacity to bind to the operator sequences (tetO) located within the promoter of the tetracycline resistance operon, and transcription is permitted.
By creating a fusion protein composed of the tetR and a potent transcriptional activator, VP16, a tetracycline- dependent trans-activating factor (tTA) was produced that retained the DNA binding and activation specificity of the tetR.
The desired regulatable gene of interest is placed under tetO plus a minimal promoter (Pmin), that contains the basic promoter elements required for transcription in all cell types. Activation of this system requires the binding of the tTA to the tetO operator sequence.
The presence of tetracycline, or other suitable ligand such as doxycycline, prevents tTA from binding to tetO and activating transcription of the gene of interest. This is referred to as the tet-off system -that is, when tetracycline is present, transcription is off. A tet-on system has also been developed, in which tetracycline induces transcription of the gene of interest.
It utilises a reverse tetracycline transcriptional activator (rtTA), designed so that it would bind to tetO and activate transcription only in the presence of tetracycline-related compounds. Doxycycline is most frequently used because it is a potent regulator in both the tet-off and tet-on systems, and can be easily supplied to mice through their water or food supply.
The tet-off and tet-on systems are binary systems-i.e., they require two genetic elements to be introduced into mice. First, a tissue-specific promoter can be used to express tTA or rtTA in a region or cell-type specific manner; then the gene of interest is inserted behind tetO and a minimal promoter. This can be achieved by creating two separate transgenic lines of mice and then crossbreeding to produce bi-genic lines.
In these lines, expression of the gene of interest may be induced by doxycycline (tet- on) or by the discontinuation of doxycycline treatment (tet- off). For example, the tet-off system has been used to investigate the effects of the transcription factor ΔFosB on psychostimulant responses.
A line of mice was generated in which expression of a ΔFosB transgene was suppressed by continuous doxycycline treatment throughout development.
Discontinuation of treatment in adult animals led to overexpression of the transgene in the nucleus accumbens and to augmentation of the rewarding and locomotors stimulant properties of cocaine. The utility of the tet-on system has also been demonstrated. For example, a line of mice was developed to examine the role of the Ca2+– activated protein phosphatase calcineurin in synaptic plasticity.
Treatment of these animals with doxycycline induced calcineurin overexpression in restricted forebrain regions, associated with deficits of neuronal plasticity and spatial learning. Rather than generating regulatable gain of function mutants with the Tet system, regulatable loss of function mutants can also be generated by combining the Tet system with the Cre-lox system.
In this arrangement, a cell type -specific promoter drives rtTA expression and Cre is linked to tetO and a minimal promoter. In the presence of doxycycline, Cre is expressed in the cell type specified by the promoter used to drive rtTA expression, and somatic cell recombination excises floxed DNA fragments in those cells-achieving an inducible cell-type-specific knockout.
In these inducible knockout mice it must be remembered that although the excision of the floxed gene can be induced relatively quickly, the appearance of any phenotype resulting from the absence of the gene product will occur gradually, depending on the degradation rate of the relevant mRNA and the half-life of the protein.
Another important limitation of strategies utilizing the Tet system relates to the inherent “leakiness” of the tetO operator; i.e., low levels of unwanted gene expression may occur during periods in which gene expression is expected to be turned off.
This may be problematic when the inducible transgene is toxic or has significant effects even when expressed at very low levels. Recent findings with tetracycline controlled transcriptional silencers indicate that it may be possible to modify the tet system to substantially reduce unwanted gene expression.