The convenience of the CreERT2 fusion is that, although the Cre will be expressed when the promoter is active (in your case the Nkx2.5... but be aware that this expression - temporal and spacial - can be very different if your Cre line is produced as a knock-in in the Nkx2.5 locus by homologous recombination or is a transgenic line using "the promoter" of Nkx2.5: in the first case it will be quite reliably follow the expression of the Nkx2.5 transcription factor, in the second case you might have quite substantial differences, depending how complete is the promoter region chosen, considering that sometimes it is impossible to reproduce a perfect promoter of a eukaryotic gene since regulatory elements can be very far away), it will not enter the nucleus, and therefore induce recombination, until the estrogen analog Tamoxifen is provided.therefore if you inject your mother at late stages of development, you will see only LacZ expression starting at that late point. To be sure your system has no leakage, also dissect and stain embryos at the same developmental age without Tamoxifen treatment.

In any case the use of the Rosa reporter line and the Nkx-Cre or of the Nkx-LacZ reporter depends on what is your aim.

If you intend to study the depletion of some gene of interest in the domain of expression of the Nkx2.5, then the first system is the best because it will tell you where your gene COULD be inactivated by the Cre line you are employing (but remember that the locus of your gene of interest might not be accessible to the Cre exactly as the Rosa locus, that's the reason of the "could"...).

If you just intend to study the expression pattern of Nkx2.5, then the fusion/insertion to/of a reporter gene will be best (but is has to be a knock-in by homologous recombination to be faithful).

Dear Dr. Boyl

Thank you for your comprehensive information about this issue. Indeed, I intend to create a NKX2.5-Lacz knock-in reporter line. For this purpose, the region between the gene promoter and first exon (before ATG) would be cut with CRISPR-Cas9 (paired Cas9 nickases) and then the cassette containing Lacz, Loxp-Neo-Loxp would be inserted in this region via homologous recombination. Left homology arm in this system is the end part of promoter (containing the proximal cis-elements) and 5’UTR of the NKX2.5 gene. Also, the right homology arm is the first exon and a part of the following first intron. After homologous recombination, the expression of reporter gene would be under control of NKX2.5 promoter. For preservation the intact translation of NKX2.5 gene, I want fuse the IRES sequence just before the first ATG in exon1. In this method, it would not be needed to detect the heterologous colonies due to two intact gene alleles.

Hi Samaneh,

I see your point. In general the strategy is fine if you need a reporter, although I would plan it differently. Translation from IRES is generally much lower compared to Cap-dependent translation, at least 5-10 times. So if your idea is to preserve the wt Nkx expression and have a reporter line to follow it, I would place the LacZ under the IRES control, not the Nkx, therefore inserting the cassette after the stop codon.

The other thing I would evaluate is if the lacZ reporter is your best option, or if a fluorescent protein might be better a better choice (for example to do co-localization studies with other cell-specific markers): although there is now a quite good anti-beta-Gal chicken Ab from Abcam for IF studies, it is not so good in cryosections, which is what you might prefer for embryonic stages, it works well only in floating sections that you can prepare for later stages embryos only and will anyway require a confocal microscope for the analysis.

Last point, expressing high levels of exogenous proteins might be toxic sometimes, so it is not a bad idea to have them under the IRES control also for this reason, if you want the homozygosity of the wt gene.

Good luck.

Thank you very much for your informative response.