Confused about cre-lox recombination system - Why is CreatorTM DNA cloning better over other cre-lox systems? (Jun/09/2007 )

I'm using CreatorTM DNA cloning Kits to express several exons of Muscular Dystrophy gene. I've read on the User manual of the CreatorTM DNA cloning kits, a number of advantages over other Cre-lox systems are listed, but i'm still not quite sure about why it states that:

This method does not require PCR cloning methods so there is no need to sequence
the entire gene insert and the sequence remains intact.

Why is other cre-lox system required PCR cloning methods? and why is this system doesn't?

Also, the kits contains pDNR-1r-Luc Control Vector, i found out that this control vector is used to monitor Cre transfer (from donor vector to Acceptor Vector) as well as to monitor expression levels in the experimental system. but how does the control vector work out that??

is the control vector insert its luciferase gene together with the gene of interest into the acceptor vector?

hmm.. actually why is it need to insert the gene of interest into Donor vector (doesn't contain promotor) and subsequently transfer the gene into various types of acceptor vectors which can express the gene as it has promotor? why can't just insert the gene into different vectors containing MCS to perform different functions?

i think my questions are kind of stupid, but i hope that someone could kindly explain to me.
sorry

---

Hyperbol and marketing language.
Just to make things clear. This system does not need any less or any more PCR cloning then any other cre-lox system. You still need to insert your gene of interest into your donor vector. How you do it is up to you. You may have to use PCR to amplify your gene before ligating it into your donor vector. You may be able to cut said gene from an existing plasmid and ligate it into Multiple cloning site in the donor vector. Or if you are luck and willing to spend money, be able to find your gene already in a donor vector in libraries that use the CreatorTM vectors.

This particular Cre-lox system is nothing special compared to other systems.




As best as I can understand the company user manual, the answer is 'no'. The luciferace is not transfered along with the gene of interest. The luciferace control vector is just a donor vector that carries a luciferace gene. If the Cre is working, the gene integrated into the acceptor vector (which contains the promoter.) Integration bring the luciferace gene close to a promoter, causing luciferace expression and causing the cell to glow green (under UV). If cells glow green then you know your vial of Cre recombinase is working. And that the loxP integration reaction should be working.



This is a technical question, concerning time, money and ability.
Consider the following situation for a moment. Lets say, you have a gene. You want to express it under the control of a strong promoter, a medium strenght promoter, a weak promoter and an inducible promoter. Sure you could clone your gene into each and every one of those promoters but it would take time. And what if your vector was very large, or your gene was large. The cloning just becomes a little bit harder.

So is there an easier way to do this? Well some people think the Cre-lox system will make things easier. Make your donor, make your acceptor , mix Cre recombinate, incubate and presto! you have your new plasmid, no need for the trials and tribulation of cloning.

Finally consider that you are a gene merchant. You are selling genes from your library to scientist. You have no idea what people do with the genes that you sell from your library. So to be just that little bit more competitive you develop a system where people can easily move your gene into the vectors that they want to use.

---