To generate a mini-horta that does not express the fluoride transporter in the rock-dissolving organ as an adult we will be utilizing the Cre/loxP system with a temporal control over the expression of Cre, so that we can, in a sense “deactivate” the expression of the loxP surrounded gene by the addition of an exogenous substance in a particular tissue. To create this model mini-horta we must create two lines of mini-horta, one with loxP sites surround the gene coding for the fluoride transporters, and another expressing the Cre-recombinase enzyme bound to a cytosolic estrogen receptor. To create our loxP mini-horta model we will utilize the protocol outlined by Claudine Kos (2004) and displayed in Figure 5-1. The “floxed” gene that we will
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We will be using the methods outlined by Claudine Kos (2004) which is displayed in Figure 5-2. We will create this model so that expression of Cre can be temporally activated in the adult stage of the mini-horta, sparing the fluoride transporter’s vital function during the developmental stages of the mini-horta. In this method outlined by Hall et al. (2009), Cre is ligated to a mutant ligand binding domain of the cytosolic estrogen receptor, so that Cre can only excise the floxed gene when tamoxifen, and estrogen receptor antagonist is applied via injection. Like the procedure with the loxP model, we will design a gene consisting of a tissue specific promotor for the tissue of the rock-dissolving organ, followed by the gene coding for Cre ligand bound estrogen receptor (ERT-Cre), such as the gene model in Figure 5-2. We will then microinject a pronucleus of several mini-horta single celled embryos with the DNA construct containing our tissue specific promoter and ERT-Cre gene. These embryos can then be implanted into a pseudopregnant female, and offspring that express Cre, assuming the gene was properly integrated should be screened for the heterozygous expression of Cre via PCR. Our Cre models only have to be heterozygous because one copy of the gene that codes for Cre is enough to produce the enzyme, unlike the loxP model which needs copies of the loxP sites on each
We will be using the methods outlined by Claudine Kos (2004) which is displayed in Figure 5-2. We will create this model so that expression of Cre can be temporally activated in the adult stage of the mini-horta, sparing the fluoride transporter’s vital function during the developmental stages of the mini-horta. In this method outlined by Hall et al. (2009), Cre is ligated to a mutant ligand binding domain of the cytosolic estrogen receptor, so that Cre can only excise the floxed gene when tamoxifen, and estrogen receptor antagonist is applied via injection. Like the procedure with the loxP model, we will design a gene consisting of a tissue specific promotor for the tissue of the rock-dissolving organ, followed by the gene coding for Cre ligand bound estrogen receptor (ERT-Cre), such as the gene model in Figure 5-2. We will then microinject a pronucleus of several mini-horta single celled embryos with the DNA construct containing our tissue specific promoter and ERT-Cre gene. These embryos can then be implanted into a pseudopregnant female, and offspring that express Cre, assuming the gene was properly integrated should be screened for the heterozygous expression of Cre via PCR. Our Cre models only have to be heterozygous because one copy of the gene that codes for Cre is enough to produce the enzyme, unlike the loxP model which needs copies of the loxP sites on each