Mittwoch, 9. Juli 2008


Half a page of experimental description can boggle your mind for an hour. No, wait... not yours - that of three biologists, two of them just starting on their PhD.

I'm talking about Nature Genetics' "Generalized lacZ expression with the ROSA26 Cre reporter strain" by P. Soriano

In short:

We have previously described a gene-trap strain, ROSA ß-geo 26, in which expression of the ß-geo reporter appears to be constitutive during embryonic development. I report here successful targeting at the ROSA26 locus and the derivation of a reporter line for monitoring Cre expression. To target the locus, a 5-kb genomic fragment was subcloned in a plasmid vector along with a diphtheria toxin (DTA) expression cassette for negative selection to produce the vector pROSA26-1. A splice acceptor sequence (SA) identical to the one used in the original gene-trap allele, a neo expression cassette flanked by loxP sites, a lacZ gene and a polyadenylation (bpA) sequence were inserted at a unique XbaI site approximately 300-bp 5´ of the original gene-trap integration site. A triple polyadenylation sequence was added to the 3´ end of the neo expression cassette to prevent transcriptional read-through. [...] Heterozygous R26R mice were bred with R26Cre mice, a general deletor mouse line made by targeting Cre to the ROSA26 locus, and embryos were collected at various stages between embryonic day (E) 8 and E16 and stained with X-Gal for lacZ activity. Embryos heterozygous for both R26Cre and R26R alleles displayed ubiquitous blue staining, whereas wild-type or heterozygous R26R embryos did not show any staining.

What is he trying to tell us? In the still-complicated-but-less-complicated words of a biologist -to-be:

Sorriano's lab generated a mouse strain in which you can monitor in which cells you successfully "knocked out" a gene.

How do you knock out a gene? Roughly, you frame the gene by a DNA-sequence called loxP. In the cells where you want to knock out the genes, you activate the gene "Cre" that will find the loxP and cut them out along with everything that's inbetween them. Tada, gene no longer there = knocked out.

Leaves the question unanswered how you activate "Cre" in only the cells where you want to knock out the genes. For that, other genes come in handy - genes that themselves, by nature, are only active in certain cells. Each of these genes will have at least one "promotor" (a sequence that starts the gene transcription) that naturally is only activated, where the gene is expressed. If you put the DNA Sequence for "Cre" behind the promotor of a gene that is expressed in only certain cells (nerve cells, for example), you can activate Cre specifically in those (nerve) cells.

So far so good. Now what did Sorriano do?

Well, he did two things. First, he put a gene (LacZ) behind a promotor that is expressed everywhere in the mouse embryo. With some help (by giving it a substance called X-Gal), LacZ can turn cells blue. So if you put LacZ behind a promotor that is activated in every embryonic cell, you can turn the entire mouse embryo blue. However, Sorriano also put in a sequence BEFORE the LacZ gene that contains a "stop" signal. So, while the promotor is activated, the transcription is terminated before LacZ is even produced.
He then flanked the STOP sequence by loxP sites, so that we have this:

[Promotor] [LoxP] [Stop] [LoxP] [LacZ].

The gene is transcribed until it reaches the stop sequence, where the transcription is terminated - before LacZ. So normally, mice with this construct don't express LacZ to help the biologist turn cells blue. These mice embryos aren't blue.

However, in cells where Cre is activated, [LoxP] [Stop] [LoxP] will be cut out and what is left is:

[Promotor] [LacZ]

Meaning, only where CRE is active, cells can be turned blue.

So far so good. Now Sorriano had to test whether this even works. For that, he put a sequence for Cre into the mouse. Smartly, he put that Cre Sequence exactly where the Promotor-loxp-stop-loxp-LacZ sequence is. That location on the mouse genome is called ROSA26.
You see, every mouse (like every human) has two copies of each gene - that is: Two ROSA26 locations (one on each sister chromosome). So, Sorriano generated mice with one ROSA26 location that held the LacZ plus stop sequence, and one that held the Cre recombinase. Since the promotor at the ROSA26 location is expressed in every embryonic cell, Cre was activated in every mouse embryo with this genetic make-up. That means every single cell expressed CRE, which then cut out the loxp-Stop-loxp from every second ROSA26 location, therefore allowing LacZ to be expressed. This means that the entire embryo could be turned blue.

Mice with two promotor-loxp-stop-loxp-LacZ sequences were lacking any active CRE and therefore could not be turned blue: There was no CRE to remove the STOP sequence before the LacZ gene, therefore no LacZ was expressed.

Mice with two Cre-sequences at the ROSA26 locations lacked the LacZ to be activated, therefore also couldn't express LacZ.

What does this serve anyone in science? Well, there are three things.

Easiest, if you want to know in which cells a certain promotor is activated, you can have Cre controlled by this promotor to simply stain all the cells where the promotor is active. You'll be learning about its genetic function, and the genetic expression of the gene that is usually controlled by this promotor.

Second, you can put your Cre behind any promotor you want (for example, a promotor only activated in liver cells). If your Cre-construction works, all liver-cells can be colored blue. Now you now know for certain that your liver-cell-only Cre works very nicely.

Third, if you then put some other genes between "loxp" sites to knock it out, cre will busily activate LacZ, but also busily knock out the gene that you want to be knocked out (a certain horomone or protein, maybe). And as a convenience, all the cells where this worked can be colored blue. And all the cells where you DIDN'T knock out your hormone or protein cannot be colored.

This system is utterly cool, though only relevant for mouse embryos.

All clear now?

Considering Sorriano had to put all this into half a page of scientific writing, it's no longer surprising that it was hard to understand.

And as a side note - the whole knock-out thing in mice deservingly lead to the inventors receiving a Nobel Prize.

Btw. Please comment and tell me where exactly I lost your attention.

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