Hi Surya I take it you mean that you inserted a gene fragment whose protein product is UV fluorescent?

I take it you also inserted a promoter along with the fragment and that the fragment has an in-frame ATG at the start of the sequence?

DNA can be read in either orientation (as it is double stranded) so if there are no frame shifts or point mutations then the protein product can be expressed (indeed in the genome genes can be I either orientation with respect to the i.e. centrosome).

If all of the above are ok then it is most likely that a failure to express the gene is either due to a) inactivation of the promoter due to methylation of the promoter in your cells (for example CMV promoter is notorious for being inactivated in some cell types such as H9C2 cells).

b) the gene has been introduced into an area of the genome that is high in heterochromatin (such as at the end of the telomeres).

Do you know where in the genome your fragment inserted? Also did you sequence the PCR product to see if any out of frame errors had been introduced?

Also how did you introduce the fragment? plasmid or retroviral?

Gary

Hi Gary, thanks for your response. Both of your points are correct - protein product of the inserted gene fragment should be fluorescent and promotor is located upsteam of the gene of interest. 

The insertion should be on the flanking region of glmS gene. The sequence of the PCR product shows no any errors. This is fine. 

Actually, I have an additional thought at the moment. The insert was excised from a high copy plasmid and finally inserted as a single copy gene in the chromosome with the help of plasmid. Could it be that lacI which is on the downstream of lac promotor (in my insert) needs to be induced with IPTG for enough expressionof the protein ?  

Hi Surya - sorry about the delay in replying to you (I've had some problem with my computer).

I guess that could be a problem as the Lacl is repressor (It has been a very long time since I used the Lac operon).

"The first control mechanism is the regulatory response to lactose, which uses an intracellular regulatory protein called the lactose repressor to hinder production of β-galactosidase in the absence of lactose. The lacI gene coding for the repressor lies nearby the lac operon and is always expressed (constitutive). If lactose is missing from the growth medium, the repressor binds very tightly to a short DNA sequence just downstream of the promoter near the beginning of lacZ called the lac operator. The repressor binding to the operator interferes with binding of RNAP to the promoter, and therefore mRNA encoding LacZ and LacY is only made at very low levels"

https://en.wikipedia.org/wiki/Lac_operon

I don't know how much of your Lacl/Lac operator is intact in your cells but it might be worth adding some lactose to your growth media and see if this helps.

However I suspect that either the gene has integrated somewhere that does not express very well or perhaps there may even be some out of frame ATG's before your gene's start codon (upstream, out of frame ATG's can reduce expression from a gene).

If you do have to repeat the generation of the cells I would personally select for stables and generate several clones from this population (I have found a great variation in protein expression from clone-clone depending upon integration site, the number of integrations per cell etc).

Sorry I can't be more help,

Gary