Gene Cloning

396 Gene Cloning
complete plants by manipulation of the make up of the media in which
they were grown. Selection of transformed cells was achieved using
glyphosate itself, since only those cells that expressed the new glyphosateresistant
EPSPS protein would be able to grow: a useful trick which
removed the need to use an antibiotic resistance marker gene in the transformation
process. Regenerating an intact maize plant from transformed
cells is an impressive, but fairly routine, exercise in cell culture, with
changes of medium needed as the regenerating tissue passes through different
stages of development.
Showing that the DNA is stably transmitted
As with the cases described above, the initial transformants made using
this process were screened to find those where expression was high and
these were then grown to maturity and back-crossed (i.e. crossed with the
wild-type parents from which the transgenic plants had first been derived).
A single inserted transgene would behave like a single dominant
Mendelian locus in this cross, and plants where this was true were identified.
Repeated back-crosses of progeny with the same wild-type parental
strain showed that even after several generations the novel gene still segregated
in an entirely predictable way.
Q12.8. Why would you expect the new EPSPS gene to be dominant in a
back-cross?
12.4 Drawbacks and Problems
Unfortunately, it is sometimes possible to give the impression that the generation
of transgenic organisms is a straightforward and trouble-free technology.
However, anyone who has spent any time working in a laboratory
where such organisms are being produced or studied would probably disagree,
and they might give the following three points as reasons.
The first is that they all require a very high level of technical skill and, frequently,
sophisticated apparatus. Manipulations such as microinjection or
plant tissue culture take months or years to learn and to perfect. There are
many points where these techniques, which after all involve manipulating
living tissue, can go awry, and anyone who is doing them routinely will have
a whole set of tricks that they use to get things to work as well as possible.
The second point is that there are many pitfalls in producing transgenic
organisms even if the initial manipulations work well. With animal transgenics,
processes such as implantation and development are ones which, even
under completely natural conditions, have a significant failure rate. The frequency
of success is further reduced with embryos that have been manipulated
in the Petri dish: microinjection, for example, is a stressful experience
for a cell and many cells will either die shortly after microinjection, or will