Creating KO mice is one of the best and advanced methods for studying the function of a gene in vivo. However, in a considerable number of cases, it has turned out to be problematic, as it can cause early embryonic lethality. Even though this is a clear indication for the importance of the candidate gene in the embryonic development, the real function of the gene in adults remains unanswered. Apart from this, inactivation of a gene in all tissues will make interpretation of the results very complicated, as it can not easily be concluded whether the phynotpe in a tissue is the consequence of gene inactivation in that particular tissue or it is due to inactivation of that gene in other tissues. To address these issues, a more advanced system has been created, by which a gene can conditionally be inactivated either in a particular cell types or in a specific time.
The Cre-loxP system has been widely used for inactivation of a gene in a particular tissue. In this system, the Cre recombinase mediates excision, recombination between loxP sites and consequently inactivation of a floxed gene or in other word a target gene flanked by two loxP sites. For creation of this system in mice two different lines of mice are required. First, a conventional transgenic mouse line expressing Cre recombinase protein in a specific tissue or cell type, and secondly a mouse line that carries a floxed gene. Crossing these two mouse lines creates a double transgenic line carrying the floxed gene and expressing the Cre protein. Expression of the cre is under the control a promoter, which directs the expression of this protein to a particular tissue or cell type. Thus, inactivation of the target gene occurs only in those cells expressing Cre. The main advantage of using this system is inactivation of a gene in a cell type-specific manner. However, similar to the conventional methods, the embryonic lethality can still be a problem.
The Cre/loxp inducible system is currently one of the most advanced tools for not only cell type-specific but also time-specific inactivation of a target gene. In this system Cre protein is fused to a mutated form of estrogen receptor (ER). In unstimulated cells Cre-ER is sequestered in the cytoplasm but after addition of the the drug Tamoxifen, which is a ligand for the mutated ER, the ER and Cre translocate to the nucleus, where Cre can excise and inactivate the gene in a particular time. See the attached film for more details.
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What happens with the endogenous gene? Assume you have the gene A and you want to delet it in Liver. You make an Albumin-Cre, but the endogenous is there.
ReplyDeleteEP
Thanks!
ReplyDeleteI am not sure if I understood your question correctly. However, If the gene A is an endogenous gene, it can be inactivated by liver-specific Cre expression. I wonder what you meant by " the endogeous is there"?
AP
This is what I meant:
ReplyDeleteAssume you want to make a tissue specific knockout in brain using a BRAIN specific promoter. (BRA) (CRE-BRA)
You want to knock-out a memory gene (MEM). Your second mice strain is a LoxP floxed MEM mouse. But what happens with the MEM gene that is already there. (Endogenous gene, not FLOXED.The mice has the MEM gene, unless you remove it by by homologeous recombination. How it is performed. In blastocytes/
EP
Hope I could understand your question this time.
ReplyDeleteThe first generation of LoxP-floxed MEM mouse are heterozygous, which have one normal MEM and one floxed MEM gene. Crossing these mice with CRE-BRA mice will generate some heterozygous LoxP-floxed MEM/Cre+ mice. Inter-crossing these mice will produce some homozygous LoxP-floxed MEM/Cre+ mice in which both MEM genes on both chromosomes are floxed.
When we treat the homozygous LoxP-floxed MEM/Cre+ mice with Tamoxifen it activates the Cre, which cuts the two floxed genes on both chromosomes.
AP