why use beta galactocidase as a positive control in co-transfection - why not an antibiotic resistant plasmid (Dec/12/2008 )
To dpo . thanks. just one more question. there is no particular sites on a plasmid for the integration of the plasmid in the genome, am I right ? so any plasmid could finally be integrated anywhere into the genome ?
That's right, you get random insertion into the genome, although I do presume that some sites (with a relaxed chromatin structure for instance) will be more prone to insertion than tightly condensed regions. You can take any plasmid you want, and in a very low percentage of transfected cells, this will incorporate in the host genome.
Beware, there are certain systems (I think the Flp-In system from Invitrogen is like this) where you use a frt site on the plasmid and a frt site in the genome and then you get site-specific insertion. But this is of course limited to cell lines already containing a frt site in their genome.
but seemingly it has a strecth of DNA that is complimentray to a fragment of DNA located on the genome downstream of the DNA for ER so i think it will incorporate here, do you think this is possible and is correct dpo?
I would think this is very unlikely. I think the region of the complementary sequence to the ER is probably limited to a couple of hundred bp? If you want site-specific insertion, you need homologous recombination, but this requires stretches of several kb of complimentary sequence in your plasmid and genome, flanking the sequence you want to insert into the genome.
is this techniques also used in knockout gene ananlysis incorporation at an exact point?
on the other hand however for our immortalisation plasmid we have no idea where this had inserted and i do not think we can tell either.
Indeed, homologous recombination is used to make knock-out cells.
The immortalisation plasmid inserted randomly and I assume your luciferase plasmid will do the same, unless you add additional sequences homologous to the host genome (but not worth the effort, imo). I would just do a cotransfection of your luciferase plasmid together with a plasmid containing an antibiotic resistance gene. In a very low percentage of cells, both vectors will insert and you will get clones resistant to the antibiotic which express luciferase, but for this you will have to test several clones.
I have used the FLP-in kit, the insertion is site specific to a FRT site, but the incorporation of the FLP-in plasmid containing the FRT site is not, so basically you get a random insertion the gene of interest into the genome, but, and this is the selling point of the kit - the insertion of your gene of interest is single copy only.
Cotchy, the most important thing you should take out of this is that the B-gal is only used as a way of estimating what proportion of your cells are transfected (your transfections are considered transient), this does not mean that the cells that have the B-gal also have the ER (BTW, this should be OR for Oestrogen receptor, otherwise people could confuse it with the endoplasmic reticulum).
If you want to stably transfect the ER(OR) plasmid then you will need to subclone the key bit of it into a new plasmid such as pCMV, with a selective marker, or as dpo says, co-transfect with a selective marker and test for clones that have luciferase activity as well as antibiotic resistance. The latter strategy is slightly more dodgy, as it is possible that the cells will drop or inactivate the luciferase gene, but retain the resistance, as the luciferase isn't being selected for directly, whereas it is being selected for directly in the former strategy.
Actually this plasmid works by expression of the SV40 large T antigen, which binds to p53 and pRB in dose dependent manner, eventually inactivating the gene. If your cells can then overcome the telomere end problem they will immortalise. You could do the same thing with E6/E7 of HSV or E1A and E1b/55K of adenoviruses.
Thanks for your advice dpo,
i am now a lot wiser on the area and understand why B gal is used as opposed to an antibiotic marker,
As b gal is not incorporated into the genome and does not change the characterisitics of the cell it is used onloy as a way of estimating a percentage of cells which have taken up this plasmid,
Which one could extrapolate to being the same number that have taken up the luciferase plasmid (although this is very unlikely to be the exact same number but for data purposes can be estimated to be the same)
I have attached the paper which describes the luciferase plasmid, it is not a paper on its development but rather a paper from a university in itlay which we colloborate with on our project,
Although it is probably not of interest to you as a away of thanking you (and bob 1 and miselle) it might contain some info that you are not aware of
They have actually developed a in-vivo detection mouse that contains a luciferase reporter gene attached to the oestrgen recpetor in every cell in the body (well every cell that already contains an oestrogen receptor) by transfection of the same plasmid we are using into a developing zygote (Fig 1 a on the paper) and so i am pretty sure it is specific for the oestrogen recpetors responsive elements in the DNA
I will talk to my supervisor (and show her these forum posts) when she returns and discuss which is the best way to proceed in terms of decding wheter to use B gal or pBabe puro as we have both systems available in the college
Again many thanks for all who left replies
Hi Bob1 thanks for your help and extra info on the pSV3-neo, i am lucky in a way that my supervisor know so much about my project which means i dont need to get bogged down on so much technical info as my background is in toxicology rather than molecular biology
She is actually developing the exact same system as my (for a post doc) only using male reproductive tissues - from primary sertoli cells which she ahs also immortalised but by using viral vectors as opposed to a plasmid
Beacuse she has always been a few steps ahead of me, i have always just followed what she has done but now i have caught up on her and we are both getting stuck on this co-transfection area
So again thanks for all your advice, i attached a paper regarding the luciferase plasmid on a previous post which you might like to read
To add apoint about the OR/ER, as with most things these days we have followed the americanisation of spelling estrogen, hence it is called ER, even in published european/british papers you will see the word oestrogen recpetor spelt with an O but then abbreviated to ER in brackets beside it,
i know this can be confusing when using the term for people who are not aware of this and i myself have mistaken ER for endoplasmic reticulum at the start of my project but it is just habit for me to do this now