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Reading from Hedgehog signaling reprograms hair follicle niche fibroblasts to a hyper-activated state:

Lineage-specific genetic tools are necessary to precisely study HF fibroblasts; however, their repertoire remains limited. Embryonic dermal condensate progenitors can be targeted with Tbx18-Cre (Clavel et al., 2012), whereas adult DS fibroblasts, with Sma-CreERT2 (Rahmani et al., 2014) and Acan- CreER (Heitman et al., 2020). DP fibroblasts activate Sox18- CreERT2 at birth (Legrand et al., 2016), Corin-Cre soon after birth (Enshell-Seijffers et al., 2010a), and CD133-CreERT2 in adult skin, albeit only with low efficiency (Zhou et al., 2016). Here, we confirm that in mouse skin, Leptin receptor (Lepr) is a faithful DP marker gene (Greco et al., 2009; Rezza et al., 2016) and show that isoform B-specific LeprB-Cre (DeFalco et al., 2001) and LeprB-CreER can be used to efficiently target postnatal DP fibroblasts. Using these genetic tools, we uncover how DP fibroblast lineage and signaling niche are regulated by the Hedgehog pathway.

I watched this video as a refresher on Cre recombinase. I attempted to Google the question with no luck. I also glanced back at one of the papers referenced. It seems it would take considerable effort for me to teach myself this answer and I'm sure there are members here who know off-hand. My understanding of Cre is that we will put it under the expression of tissue-specific promoters, as is stated in the video. I covered this technique in a paper I read last semester. I assume ER stands for endoplasmic reticulum, possibly? Where additional confusion is arising for me is that in the sentences describing "at birth" and "soon after birth" it seems in the way it's presented that these Cre could be native to the organism but we add Cre to the genome in all these cases, right?

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  • $\begingroup$ FWIW, to find a reference to include in my answer I searched "CreER cre recombinase" (to avoid false positives with "creer") in Google and found an example paper to cite immediately. Perhaps it helped that I'm familiar with the system and knew what I was looking for. $\endgroup$
    – Bryan Krause
    Jul 6, 2022 at 16:47
  • $\begingroup$ Your answer is absolutely helpful, thank you for typing it up and for the quick reply. I definitely am in a much better position to complete the investigation myself if need be. Instead of accepting your answer, I am going to leave the question open as while your answer is a start there is quite a bit going on in that paragraph I quoted from the paper. $\endgroup$ Jul 6, 2022 at 16:51

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CreER is Cre coupled with a binding site for the estrogen receptor ("ER") to make an inducible version of the Cre system triggered by tamoxifen. CreERT2 is just a particular variety of this system with some mutations that improve the function.

Feil, S., Valtcheva, N., & Feil, R. (2009). Inducible cre mice. In Gene knockout protocols (pp. 343-363). Humana Press.

Some quotes from this paper:

To add inducibility to the Cre/lox system, ligand-dependent chimeric Cre recombinases, so-called CreER recombinases, have been developed (3–6). They consist of Cre fused to mutated hormone-binding domains of the estrogen receptor. The CreER recombinases are inactive, but can be activated by the synthetic estrogen receptor ligand 4-hydroxytamoxifen (OHT), therefore allowing for external temporal control of Cre activity.

The properties of CreER recombinases were continuously improved to decrease the background activity in the absence of inducer and to increase the sensitivity and efficiency of tamoxifen-induced recombination in mice. The CreERT2 recombinase, which contains the human estrogen receptor ligand-binding domain with a G400V/M543A/L544A triple mutation, is currently the sharpest tool in the CreER box and its use is highly recommended for inducible mutagenesis in the mouse (4 , 9 , 10).

Briefly, the two common ways to use the Cre-Lox system these days are either 1) You have a gene you want to knock out in only certain cells; you've modified this gene to be surrounded by LoxP sites (often referred to as 'floxed' as a shortening of 'flanked by LoxP sites'), or 2) You have some genetic construct you want to express in only certain cells (for example a reporter like green fluorescent protein/GFP), so you start with an inserted gene with a 'floxed' stop codon that comes before the GFP.

You then express Cre under a desired promoter (let's call it YourFavoritePromoter), Cre cuts out the floxed section: either the gene to conditionally knock-out (1), or the stop codon so your GFP is produced (2), but only in cells that have YourFavoritePromoter expression.

However, this is a permanent change in a lineage. If Cre is ever expressed at any time during a cell's lineage, all subsequent daughter cells will have had the floxed part removed. That means the cells you mark with GFP are not necessarily those expressing YourFavoritePromoter now, but cells that sometime in the past expressed YourFavoritePromoter.

You can use CreER to get around this, as Cre will only be expressed with tamoxifen present. That allows you to control the time window in which Cre is expressed, and therefore the time window in which you are identifying cells with YourFavoritePromoter.

You'd want to follow up with all the individual references in your paragraph to see what they are doing specifically. For example, in the "Legrand et al 2016" paper:

Sox18 is expressed specifically in the DP in all dorsal hair follicle types; however, at birth its expression is restricted to zigzag hairs lacking Sox2 expression in the DP (Driskell et al., 2009, Pennisi et al., 2000)

So, if you want to tag "zigzag hairs" (I really have no idea what these are, but it doesn't matter for understanding the system), you use Sox18-CreER and give tamoxifen at birth. If you used ordinary Sox18-Cre, you'd label "all dorsal hair follicles" instead.

All of these are artificially introduced constructs. A frequent method for efficiency is to have some homozygous mouse strain that expresses the gene you want to see (say, floxed GFP in our example), and a different mouse strain that has the Cre construct you want (say, Sox18-CreER). Each are created using standard transgenic techniques; many researchers don't need to bother with those details because you can just buy the mice, but if you want a particular unique line you have to make them. You then breed them together to get mice that have Cre and the floxed gene.

Here's what they did (quotes from Legrand et al):

Make mice with floxed version of gene they want to control expression of (STAT5A/B; note here they're actually doing a conditional knockout, so rather than have Cre remove a stop codon it's removing the entire gene):

STAT5A/Blox/lox mice (Cui et al., 2004) were first crossed with Flash/+ reporter mice to generate mice homozygous for the STAT5 floxed allele containing the previously described Flash Wnt-reporter transgene (Hodgson et al., 2014)

Breed these with the Sox18-CreERT2 mice so they have tamoxifen-controllable cre expression under the Sox18 promoter (extra complication here that these mice also express a reporter gene; that one works with the stop codon method I described above):

These mice were then crossed with B6;129S-Sox18tm1(GFP/cre/ERT2)Pzg/J (Sox18GCE/+) mice to generate Sox18GCE/+ mice homozygous for the STAT5A and STAT5B floxed alleles, although also containing the Flash reporter transgene

Give tamoxifen at birth to express cre in a specific population of cells which will knockout STAT5A/B and express the GFP reporter.

Recombination in these mice and their appropriate control groups was induced at birth using a single dose of 1mg tamoxifen.

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    $\begingroup$ Thank you, Bryan, for your well-thought-out answer. My only suggestion would be to define acronyms before you use them (I think I also made this mistake in some of my earlier content). I know what GFP is but it's about making it easier for the next guy. Anyhow, I definitely feel up to my neck with this paper and am going to pause, step back, and read the chapter you referenced. Where I struggle is with visualization of the crossing of mice, and like anything needs practice. I am open to any specific literature (or exercises) you feel may be helpful before I continue reading this paper. $\endgroup$ Jul 6, 2022 at 19:25
  • $\begingroup$ @neurosciencecalc I think it'll come with experience. Crossing mice is just like Mendel's experiments with pea plants, I'd go re-read those sections of a biology textbook for a refresher. $\endgroup$
    – Bryan Krause
    Jul 6, 2022 at 19:26
  • $\begingroup$ Sounds good. Thank you, Bryan. $\endgroup$ Jul 6, 2022 at 19:28
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    $\begingroup$ I feel kind of silly for my question now that I am understanding more, but that's all part of the learning process. I see now that the notation is 'tissue_specific_promotor-CreERT2' and you did such a great job explaining the CreERT2 and that chapter you linked to is great as well. Thanks again for your help. $\endgroup$ Jul 6, 2022 at 21:39

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