hey,
I have generated stable cell lines with random entegration of pCMV-miR vector.
Vector contains a neoR gene fused with GFP with IRES.
I got some GFP+ and GFP- colonies.
and GFP- colonies shows an over-expressed profile for my miRNA while only %70 of GFP+ colonies are over-expressed for miRNA.
During genomic entegration the plasmid can be cutted randomly. Thus, it may breake my miRNA sequence, neoR gene as well as GFP sequence.
GFP+, Gene expression negative cells are probably cleaved from miRNA gene casset. I am okey with it.
GFP+, Gene expression possitive cells are probably claved from origions of vectors. I am okey with it.
GFP- but Gene expression possitive cells are probably claved from GFP sequence. In this point NeoR gene can only be expressed withot PolyA signal. Can this casset be expressed biologly functionaly ?
so in brief can a gene be expresed without any PolyA signal in a normal plasmid system not lentiviral.
my vector is attached.
Thanks for answers
Poly A tails stabilize mRNA preventing its degradation and aid in its export to the cytoplasm for translation. Although the poly A signal placed on your construct may be cleaved off during integration, the flanking sequence at the integration site might happen to contain such a signal sequence. So, without knowing the sequence of your RNA, it's not certain that your RNA does not have a poly A tail.
Ah, you are right; where it integrated to genome may contain a Poly A signal. Thanks
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