Consider, that the ablation of this particular gene is lethal for mouse embryonic development. There is no way to use CRISPR/Cas9? I was thinking about Cre-LoxP! Please kindly grant me your suggestions and critics.
Dear Saeid
Hello,
If you are eager to use Cre/ LoxP system, there are two most common models:
1. The tetracycline/doxycycline binary trans-activation model
Tetracycline (Tet)-inducible systems use an artificial protein (tetracycline- responsive trans-activator (tTA)) to regulate the expression of Cre proteins. The main disadvantage of this so-called Tet-off system is the high toxicity of Tet/Dox when used in the long-term treatment of mice. To circumvent this problem the “Tet-on” system was developed, where a reverse tTa (rtTA) protein only binds to the DNA upon binding to Tet/Dox, which then results in activation of Cre expression. The advantage of this method is that mice do not have to be treated
continuously with Tet/Dox. In addition, expression of the Cre recombinase solely depends on saturation of the drug in the tissue. Using cell/tissue-specific promoters to drive tTA/rtTA fusion protein expression allows spatial control of the turning off or on of genes in the tissue or cells of interest (Jaubert et al., 2004).
2. The tamoxifen–Cre–inducible model
The system is based on nuclear hormone receptors that translocate into the nucleus to regulate gene expression when bound to their corresponding hormone ligand. In the inactive state the estrogen receptor (ER) is bound to heat shock protein 90 (Hsp90) and is thereby excluded from the nucleus. When estrogen or tamoxifen binds the hormone binding site of the ER, Hsp90 is released and the ER shuttles into the nucleus. Upon application of tamoxifen, the Cre–ER protein is released from Hsp90 and can now enter the nucleus, where Cre recombines the loxP-flanked (floxed) target gene Garcia and Mills, 2002; Lewandoski, 2001; u et al., 1994; Denton et al., 2009).
You can also use CRISPR system to achieve conditional KO/ KI models.
The inducible CRISPR (iCRISPR) system can be used effectively to create biallelic mutation in multiple target loci and, thus, provides a flexible and fast platform to study loss-of-function phenotypes in vivo. The doxycycline-regulated Cas9 induction enables widespread gene disruption in multiple tissues and that limiting the duration of Cas9 expression or using a Cas9D10A (Cas9n) variant can regulate the frequency and size of target gene modifications, respectively (Dow et al., 2015).
Moreover, you are able to generate inducible gene knockout (iKO) hPSC lines by combining CRISPR/Cas9-mediated genome editing with the Flp/FRT and Cre/LoxP system. In this two-step strategy, ‘‘dual sgRNA targeting’’ is essential for biallelic KI of FRT sequences to flank the exon and you should develope a strategy to simultaneously insert an activity controllable recombinase-expressing cassette and remove the drug-resistance gene, thus speeding up the generation of iKO hPSC lines. This strategy is applicable for developmental genes such as SOX2, PAX6, OTX2, and AGO2 (Chen et al., 2015).
You can also use genome editing tools to create conditional KO Mouse Embryonic Stem Cells (mESCs) in a single-step strategy. In this protocol you should develope a reporter system to enrich for cells with engineered nuclease-assisted HR events. You will achieve single-step biallelic and seamless integration of two loxP sites for Cre recombinase-mediated inducible gene knockout, as well as biallelic endogenous gene tagging with high efficiency. As the authors claimed; their approach reduces the time and resources required for conditional knockout mESC generation dramatically (Flemr et al., 2015).
Good luck,
Dear Hadi,
Good day!
I found it a very well-organized answer and straight to what I was in need to know! This way, I can sketch the trajectory of the research accurately.
Thanks to your time.
Sorry, I have no expertise in the field. Hadi"s answer seems ok.
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