Reporter genes

Andrew John Millar ajm2m at FARADAY.CLAS.VIRGINIA.EDU
Thu Oct 6 18:18:14 EST 1994


Dear DopsNet,

Here are some starting points for answers to reporter gene
questions, especially luminescence and Luciferase:

Bronstein et al. (1994) Analytical Biochemistry 219 pp 169-181.
[A review of reporter genes for which luminescence assays are
available.]

Firefly luciferase (LUC) stability:
        Thompson et al. (1991) Gene 103 pp 171-177
        [Stability assessed in mammalian cell cultures, 
        Half life of CAT activity 50 hours, LUC activity 3 hours]

LUC in plants:
        Schneider et al. (1990) Plant Mol. Biol. 14 pp.935-947 
        [several parameters involved in LUC assays in vivo,
        using rbcS and 35S::LUC in tobacco]

        Millar et al. (1992) Plant Cell 4 pp 1075-1087
        [Northerns, Westerns, LUC activity in extracts
        and in vivo, in CAB2::LUC and 35S::Luc tobacco]

        Luehrsen et al. (1992) Meth. enzymol. 216 pp 397-414
        [Analysis of LUC activity in plant extracts-
        N.B. This whole volume is devoted to reporter genes]

        Millar et al. (1992) Plant Mol Biol Rep 10 pp 324-337
        [protocols for luc assays in vivo, including an example 
        of the dramatic effect of luciferin on LUC activity in vivo
        (in Arabidopsis). Also check article on bacterial 
        luciferase in same issue]

        and many other studies that use the reporter, including
        Anderson et al., this month's Plant Journal.

We (Steve Kay's lab) have used the firefly luciferase to follow
circadian regulation of CAB transcription in vivo. We have not
measured the stability of the protein or activity directly, but
our data is consistent with a half life of 2-3 hours at most, for 
LUC activity in cab:Luc transgenics that have been pre-treated
with luciferin.

Cheers,
Andrew






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