Cloning worked, but the expression vector is weird - (Apr/19/2014 )

So for 2months now I have been trying to clone a gene of interest into a vector.

All the steps (PCR-amplification, purification, digestion, electrophoresis) worked well. And even the final ligation, but then the overnight-culture of the plates never gave me colonies!!! But this time I got the colonies!!!!

So now I need verify whether the insert really is in there and in right orientation. That will be done by performing a RE-digest and hopefully I will get fragments of the correct size that I expect. Then we will also send it in for sequencing, to make sure there are no mutations in the coding sequence of the gene.

 

But then the next obstacle is the over-expression study. And I just don't understand why my supervisor gave me the task of clonig the insert into the vector at the position it now has been done:

- The insert is located immediately downstream of a long PolyA-sequence.

- There is no promoter at all coupled to the insert (only a ATG start-codon)

- The only promoter I have is the T7 and that is located on the other half of the circular vector

 

So I don't understand how we will be able to use this construct as a OVER-EXPRESSION study. Unless we yet again sub clone the insert to a new expression-vector. And that would just be counterproductive and waste of time since it took me 2 months to clone the insert to where it is now!!!!!!

I can't just inject this vector into the mouse and hope that it will be constitutively expressed. So what am I missing here?

Which vector is it?  It doesn't sound like an expression vector, unless you are planning on doing some RNA transcription and injection sort of thing.

It a vector he has designed on his own. It has the following:

MSC

Amp-resistance gene

a set of different fluorescent reporter-genes

T7 promoter

lac promoter

 

But even if the aim is to do RNA transcription and injection, the gene still has to be transcribed. And how can that be when there is no upstream promoter to the gene?

 

bob1 on Sat Apr 19 08:50:47 2014 said:

Which vector is it?  It doesn't sound like an expression vector, unless you are planning on doing some RNA transcription and injection sort of thing.

The T7 is for transcription - this needs to be on the reverse strand to produce a RNA that is the same as the sequence you want.

 

I don't think this one will work for over-expression unless it is running off the lac promoter, you would have to subclone into an appropriate vector.

1) What do you mean with "this needs to be on the reverse strand to produce a RNA that is the same as the sequence you want."

​Doesn't the T7 promoter need to be immediately upstream of the gene and running in the same 5' - 3' direction on the upper strand?

Why do you mean it should be running on the reverse strand in 3' - 5' direction? (if that is what you meant?)

 

2) Also, I asked my supervisor why there is no promoter controlling the inserted gene. And he said that we don't need one. Instead the insert is immediately upstream/under control of a beta-glob intron-sequence. And that is only because to give the gene stability he told me.

And now we go to the next step and inject the construct into our rats.

 

But I don't get it. If we want to study over-expression, then:

- we NEED a promoter controlling the gene

- also, what is the point in having a beta-glob sequence as a stability-enhancer when the gene will NOT BE TRANSCRIBED/TRANSLATED?

 

I understand that a 5' UTR sequence and a polyA-sequence at the 3'UTR end of any mRNA/protein is important and function by preventing the coding sequences of mRNA/protein being degraded while translocating into the cytoplasm from the nucleus in order to be translated into protein.

But this just doesn't make sense to have that in a GENE-sequence (and not mRNA/protein) that is not even under the control of any promoter.

 

So even if we inject it into our rats, how is that gene even gonna be expressed, let alone studied/evaluated?

 

bob1 on Tue Apr 22 02:14:10 2014 said:

The T7 is for transcription - this needs to be on the reverse strand to produce a RNA that is the same as the sequence you want.

 

I don't think this one will work for over-expression unless it is running off the lac promoter, you would have to subclone into an appropriate vector.

T7 polymerase is often used to produce sense and/or antisense RNA for in vitro transcription...

 

The B-globulin sequence seems like it is acting as a promoter style sequence or perhaps an inducer.  I don't really know enough about this sort of thing though.  I'm just used to basic plasmids with viral promoters.

OK. still sounds weird to have no promoter at all. And even if the beta-glob intron would act as an inducer, it still can't be possible to initiate gene-trancription since the lac-promoter is on the other end of the circular construct. There would be impossible to initiate physical interaction between the inducer and lac-promoter.

 

And, my supervisor specifically said it only acted as a stabilisator.

 

But still, thanks for the help and comments.