In a recent study (Deluna, 2008) it states that Mouse models are the predominant animal model used currently for research in disease. Several qualities of mice account of their attractiveness as models for studying disease. Mice are among the smallest known, with the average adult weighing 20-40g, roughly 2500-fold lighter than humans. Mice also have short time to sexual maturity, approximately ten weeks, breed proliferically and have roughly five to ten pups per litter. Finally, most strains of mice used in laboratory settings are docile and easy to handle. Thus, large numbers of mice can be rapidly and easily generated and maintained, a property useful for large - scale experiments with drugs or for studies in genetics. (Deluna, 2008)
As schizophrenia is the disease of the brain and the receptor X is only expressed in the small subset of neurons, therefore in order to find the function of these cells the gene that caused the receptor X to be produced should be known. If this is known, we will be able to produce a Transgenic Mouse. Knock-out mouse is a laboratory mouse, which an existing gene has been inactivated, or "knocked out", by replacing or disrupting it with artificial piece of DNA. The loss of gene activity often causes changes in a mouse's phenotype, which includes appearance, behavior, and other observable physical and biochemical characteristics. Knocking out the activity of a gene provides valuable clues and what the gene normally does. Humans share many genes with mice. Consequently, observing the characteristics of knockout mice gives researchers information that can be used to better understand how a similar gene may cause or contribute to disease in humans.
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In order to identify the function of the cells that express receptor X and how discovery of new anti-schizophrenic drugs active against the human receptor can be achieved in vivo, is through a transgenic animal. In order to accomplish this a tissue specific knock out transgenic animal has to be produced. Housekeeping genes are expressed in all types of cells at all stages of development, other genes are normally expressed in only certain types of cells when turned on by the appropriate signals (e.g arrival of a hormone). However, it turns out that genes that are only expressed in certain adult tissues may nonetheless be vital during embryonic development. In such cases, the animals do not survive long enough for their knockout gene to be studied. () Fortunately, there are now techniques with which transgenic mice can be made where a particular gene gets knocked out in only one type of cell. One type of such model is the known as the Cre-Lox Technology model. As conditional and genetic deletion, somatic cell mutagenesis, embryonic lethality in many conventional gene knockouts impede attempts to study gene function in older animals. As Riding, Ryding, Sharp and Mullins (2007) stated that one way of avoiding this is to ablate specific genes at later stages of developmental or adulthood using recombinases. Cre is one the two members of the alpha-integrase family of site specific recombinases, has imporved invaluable for conditional transgenic use. Cre causes recombination between Lox P (Locus of crossover) sites. Its function is to maintain phage encoding plasmids as monomers. Therefore the only requirements for DNA rearrangement are the integrase and the recombinant sites: no additional cellular factors are necessary. Lox P sites are 34 bp DNA sequences comprising two 13bp palindromes separated by an asymmetric 8 bp core. The core region is responsible for the directionality of the Lox P sites. The recombinase catalyses the DNA strand exchange between the two aligned recombination sites, resulting in deletion, duplication, integration, inversion or translocation of sequences, according to the orientation of the recombination sites and the number per molecules involved. (Ryding, Sharp, & Mullins, 2007)
In order for the Cre-Lox principle to work, a Lox P mouse and Cre mouse has to produced. Therefore the Lox P mouse which contains the cells carrying the endogenous gene for receptor X will have Lox P sites flanking on either side of the gene. The Cre mouse that will contain the Cre trangene, the mouse is heterozygous for the gene X knockout. Therefore by mating the Cre mouse and Lox P mouse, the Cre-Lox P Trangenic Mouse is produced. The cells that have the gene that produces receptor X will be the only cells that have expression for Cre. This therefore allows the expression of our gene in interest. Therefore only cells that have Cre and Lox P excision will occur. In all other cell types there is no Cre expression thus our gene function remains the same. This is known as tissue specific gene activation. ( (Rao & Monks, 2009)
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Several different systems are now available to regulate the expression of transgenes using external inducers. Tertiary systems have bcome the inducible regulatory systems of choise. Of these the most widely used, and therefore validated, is the tertacyline system, which has been employed in several studies. Recently, ecdysone systems have become the inducible regulatory systems choice. Of these, the most widely used, and therefore validated, is the tetracycline system, which has been employed in several successful studies. (Tornell & Snaith, 2002)
A transgenic mouse will be produced that expresses cre recombinase only in the subset of neuron cells that express receptor X and only following tetracycline treatment. This spatial temporal specificity will be achieved by two trangenes. The first transgene will be the human receptor X (that causes schizophrenia) promoter to drive expression of the reverse tetracycline - controlled activator (rtTA). The secons transgene will be the one of tetracycline responsive promoter to drive expression of (recombinase). Therefore a tetracycline response system can used, which will consist of:
The tetracycline repressor protein (Tet R) fused to the herpes simplex virus VP16 transcriptional activation domain.
The tetracycline operator sequence (tetO) linked to a minimal promoter element that controls transcription of a downstream gene.
Binding of TetR-VP16 fusion to the tetO sequences activated transcription. .
Uses a mutant variant of TetR-VP16 transactivation protein, rtTA presence of tetracycline/doxycycline induces gene expression. (McLean, 2011)
Therefore we can use a system in which the cre recombinase is controlled by the Tet Opron. Therefore when doxycycline/tetracycline is given we will able to see expression of the transgene and is only seen in the small subset of neurons and that Cre expression in the absence of tetracycline is neglible. In this way Cre is expressed throughout development by allowing Cre expression to begin at later developmental stages. This temporal control of transgene expression has several applications, including overcoming embryonic or perinatal lethality due to transgene expression. The activation of transgenes can be provided by the reverse tetracycline-controlled transactivator (rtTA) system. The rtTA transcription factor requires tetracycline, or its analog doxycycline (Dox), to bind tetracycline response elements (TRE) and thereby promote gene transcription. TRE can effectively drive gene expression in mammalian cells when used in conjunction with the minimal human cytomegalovirus (CMV) promoter. This regulatory system is also known as the Tet-On system. Therefore when tetracycline is implied and it enters the cells, it binds to response elements upstream of the cre recombinase gene on a trangene under the control of an appropriate promoter. Tet binding results in the activation of Cre expression and subsequent binding Lox P sites located on the target gene of interest. (Rao & Monks, 2009)
A transgenic mouse in which cre is induced/non-induced by tetracycline/docycline, will be able to give us the function of the cells. If testing new anti-schizophrenic drugs active against the human receptor with and without tetracycline can aid in the discovery of the best schizophrenic drug. Although this Tetracycline induced cre system is very useful , the most widely used system today is the tamoxifen - induced LBD-cre fusion protein. In which a ligand binds to cell surface receptor. The ligand -receptor complex is internalized and binds to Cre fusion protein. Ligand - LR - cre complex translocates to the nucleus and binds to the Lox P sites. Therefore LoxP mediates recombination. In this case the ligand that would activate the transgenes would be Tamoxifen. This would control not only the site of recombination but also timing. Several strategies have been designed to address this, using inducible forms of cre recombinase fusion of ligand binding domain (LBD) of a mutated oestrogen receptor to cre results in a hybrid that is active in the presence of tamoxifen. Ligand activated cre shows great promise for controlling time of recombinases. (Rao & Monks, 2009)