Transgenic reporter constructs
rkoedood at bio.bu.edu
Wed Aug 28 13:41:30 EST 1996
Here are the 'rules' we heard about when making our transgenic constructs:
the vector backbone is 'bad', undesirable (toxic? forgot the reason). Including
and intron increases expression. Transgene should be linearized.
Besides that, most important is the tissue-specific expression of your
trasncription control elements you include in your transgene. However, we
found that expression patterns in tissue culture doesn't necessarily reflect
tissue specificity in the embryo (see our paper on CMV promoter/enhancer,
Koedood et al, 1995, J. Virol. 69, 2194-2207).
Another point to keep in mind is that the site of integration may influence
the expression (in most cases by silencing it). So, to make conclusions
about effects of your transgene, you should have several, independent
lines in which the transgene is expressed.
You may want to look into adding something like an LCR (locus control region,
though note that they often (or always) aretissue specific themselves) or
SAR sequences, to reduce chromosome silencing effects.
milstone (milstone at rascal.med.harvard.edu) wrote:
: The general dogma is that "vector" sequences can (that is, have
: occasionally been observed to) alter the specificity of expression
: driven by particular regulatory sequences in transgenic mice. My
: impression is that the effect is quite variable with different
: regulatory elements and that few investigators are currently interested
: in systematically studying this aspect of transgenesis. Founders with
: faithful expression can definately be generated at high frequency
: without incorporating "protective" cloning vector flanking sequences in
: transgenic constructs. It seems to me that the potential benefits of
: including extra sequence are not very great.
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