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has in fact been incorporated into the germ line. Because transformation meth-
ods are relatively inefficient, a screening method is needed to rapidly and con-
sistently identify transformed individuals. This process is relatively simple in
Drosophila
, where a wealth of genetic information and visible markers, such as
eye color genes, can be used to identify transgenic individuals. Many pest and
beneficial arthropods lack such extensive genetic information or markers. It is
possible to use specific primers and the PCR to confirm that the DNA sequences
are present, but this only indicates the DNA is present—not that it has been
incorporated into the genome. Southern-blot analyses can confirm the integra-
tion and number of integrations. Ideally, the insertion site of the gene can be
determined by inverse PCR and sequencing to obtain flanking sequences, as well.
Identifying transformed individuals also could be achieved using a pesticide-
resistance gene as a selectable marker. However, the release of pesticide-resis-
tant pest arthropods into the environment would create concerns about risk.
Researchers working with the Medfly resolved to forego using resistance genes
as selectable markers for this reason (
Ashburner et al. 1998
). Concerns about
releasing pesticide-resistant natural enemies might be lower, except that there
is a possibility of HT of pesticide resistance genes from beneficial to pest species.
No one knows at present how to quantify this potential risk.
Another option is to use antibiotic resistance genes as selectable markers to
identify transgenic arthropods. However, HT of antibiotic resistance genes into
microbes in the environment could result in increased levels of antibiotic resis-
tance in pathogenic microbes. Antibiotic resistance genes are no longer con-
sidered safe for release into the environment in transgenic crops and methods
have been developed to remove them (
Yoder and Goldsbrough 1994, Witte
1998, Ebinuma et al. 2001, Matthews et al. 2001
). It is probably desirable to
eliminate unneeded marker genes from arthropods before their permanent
release into the environment.
Another potential marker is based on the
β
-galactosidase gene (the
lacZ
con-
struct) isolated from
E. coli
, which can be detected by an assay that produces a
blue color in the transformed insects and mites. This construct has been pres-
ent in a number of organisms released into the environment and risks associ-
ated with the release of this construct are considered low (
Hoy 2000a,b
). Eye
color (
Zwiebel et al. 1995
,
White et al. 1996, Cornel et al. 1997
,
Ke et al. 1997
,
Berghammer et al. 1999, Sarkar and Collins 2000
) and red or green fluorescent
protein genes (
Higgs and Lewis 2000, Horn et al. 2000, Pinkerton et al. 2000
)
also are considered safe selectable markers if transgenic arthropods are to be
released into the environment. Unfortunately, transgenic arthropods with
