Generating Transgenic Mice

[enemiimene]

I am reading: "Charaxterization of Astrocyte-Specific Conditional Knockouts" by Casper et al. I have trouble understanding how they generated their transgenic mice. They used a Cre-reporter Recombinase. I would appreciate it if one of you referred me to where I can find more information on how to generate transgenic mice and specifically the method they used.

Just do a google search and it will show up. I can't post url's yet.

please skim through the materials and methods and provide me a good explanation/summary in baby steps because my background in mice genetics is minimal.

Thank you ahead of time.

[knockout maker]

Hi enemiimene,
Do not worry if you have problems understanding the mice business. I bet more than 90% of the principal investigators older than 40 and doing mice at their laboratories do not understand neither how does it work, but just let their PhDs to struggle with it.
Let´s start. The researchers in this paper wanted to express Cre recombinase specifically in astrocytes. In order to do that they use an Astrocyte specific promoter such as hGFAP (meaning a piece (2kb-10kb) of the original promoter of that gene) to drive a tet transactivator. So this transactivator will be exclusively expressed in astrocytes (or at least that is what they think). Secondly, you need a separate transgene with a CMV (ubiquitous) promoter driving the expression of Cre recombinase, but with a tet operator in between, to which the transactivator will bind and do its job. As the system they use is a tet-off system, the transactivator will bind and activate the expression in normal conditions , unless doxyciclin is added to the system. In other words, the transactivator, exclusively express in astrocytes will drive Cre expression in this cell type after doxycicline addition, but in the rest of the organism there will be no Cre recombinase as the transactivator is not present because the GEFP promoter will limit the expression to this cell type. Got it?.
Another strategy is based on specific expression of Cre recombinase in Astrocytes: instead of a double transgenic squeme, one just needs a single transgenic mice here. The strategy is simply to place an inducible version of Cre recombinase (CreERT2 for example, which mantain Cre recombinase cytoplasmically located unless tamoxyfen is present, which translocates Cre into the nucleus) under the astrocyte specific promoter GFAP. So in principle Cre should be expressed and do this job just in astrocytes, and will be inactive unless tamoxifen is present. Thye same applies to the progesterone-cre fusion and merCremer.
The reporter mice they generated (ACR mice) consisted in a knock-in at the ROSA26 locus, placing a sort of stop cassette in between the GFAP promoter and a reporter, in this case they use egfp as a marker to see how good the strategy was. So that gen downstream the promoter is not expressed unless Cre is specifically expressed in astrocytes (following the above mentioned strategies), because there is an stop cassette (in that case it is a LacZ, so one can monitor non-excised cells) flanked by loxP sites: after Cre expression the stop cassette will pop out and thus will allow the expression as Cre will recognize the loxP sites and excise the cassette. These types of stop cassettes use to have multiple polyadenilation signals in order to ensure transcription is stopped. After excision, as the promoter is astrocyte specific, the gene will be expressed just in astrocytes.
In general, reporter lines are mice with reporters knocked into the ROSA26 locus, which is a locus with four main features: highly and ubiquitously expressed, easy to target, highly accesible to Cre recombinase and does not affect the mice (there is no phenotype in homocygosis). Therefore these mice are useful tools to check how efficient Cre recombinase activity was, and that s why the authors use them to check which of the strategies is superior.
Any other question?.
Cheers!!

[amicia]

good explanation, but in your review did you forget the news approches, perpahps are you referring only to the paper on the desk?