homolgous recombination / gene targetti

mjordan at pbi.lan.nrc.ca mjordan at pbi.lan.nrc.ca
Thu Oct 29 11:26:00 EST 1992


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Ed Rybicki writes:

>This is a plea for help with literature and / or info:  I would like some
>kind soul to tell me - briefly - whether much is known about homologous
>recombina- tion / gene targetting in plants.  Specifically, I would like to
>know whether anyone has tried (or knows of someone who has) to introduce
>foreign genes into plant chromosomal DNA by means of homologous
>recombination.  By this I mean the mechanism that is used to get things into
>E coli chromosome, poxviruses, baculoviruses, etc - that is, putting a
>gene/promoter construct in between a few hundred bases which have homology to
>the ends of a gene which is normally to be found in the genome to be
>engineered.

>Does anyone know of an instance where this has been done for plant
>chromosomal genes?  Which gene(s)?  How much overlap was necessary?  Does it
>work the same in dicots and monocots?

Ed- The only cases of gene targeting I am aware of are those of Paszkowski et
al. (1988) EMBO J.:4021-4026 and Lee et al. (1990) The Plant Cell:415-425. In
the former case plants were transformed with a nonfunctional NPTII gene and
were retransformed with a vector carrying the missing part of the gene. By
using kanamycin selection transformants were obtained as a result of
homologous transformation. The second paper is similar except a
nonfunctional fragment of the acetolactate synthase gene (containing
a mutant site conferring herbicide resistance) was used. Upon insertion into
tobacco herbicide resistant transformants were obtained indicating correction
of the endogenous gene to the resistant form. In both studies the ratio of
homologous events to random events was 10-4. Clearly not in a useful range
especially if no selection method is available. I would be very interested if
anyone knows of other studies where the efficiency was increased. T-DNA
integration seems to be very much a random process.

Mark Jordan
mjordan at pbi.lan.nrc.ca
National Research Council
Plant Biotechnology Institute
Saskatoon, SK
Canada



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