Biomedical Engineering Reference
In-Depth Information
known as Cre recombinase-mediated cassette exchange (RMCE), which targets the
transgene into a predetermined chromosomal integration site. Second, we describe the use
of the chicken B-cell line, DT40, for the generation of microcell fusion for the homologous
recombination modification of genomic regions maintaining the chromosome integrity for
subsequent studies.
7.2 Methods and approaches
7.2.1 Site-specific chromosomal integration in mammalian cells
Owing to the complexity of regulatory elements, their study requires the comparison of
different portions and/or the analysis by point mutations in a chromatinized context. How-
ever, random chromosomal integration in a number of uncontrolled sites and transgene
copies generally cannot be predicted or reproduced with precision because of chromoso-
mal position effects. The inability to control the site of integration, the number of integrated
copies and the level of expression of transgenes has impeded progress in studies of both gene
expression and the physiological effects of transgenes. The RMCE Protocols 7.1 and 7.2 rep-
resents, when cell lines are used, a direct and reproducible strategy for transgene integration
to predetermined chromosomal loci through the use of sequence-specific Cre recombinase
[29-31]. This method is based on the establishment of independent, single-copy integrants
of the stably transfected pL1-HYTK-L2 vector containing the
CMV-HYTK
gene. These can
subsequently be used for the generation of recipient stable cell lines enabling positive selec-
tion with hygromycin. Once characterized by Southern blot and single-copy integrants are
confirmed, they can be subjected to a negative selection with gancyclovir to select those
cells where the cassette exchange has occurred. This protocol relies on the use of two
opposing
loxP
sequences which induce cassette inversion and test cassette exchange (see
Figure 7.1a). Cassette exchange will depend on the efficiency of Cre recombinase expres-
sion and probably the genomic site of integration. It is worth mentioning that the cassette
would integrate in one orientation in half of the clones and in the reverse orientation in the
other half.
One central aspect of RMCE methodology is that, by determining the genomic inser-
tional site, we can eliminate chromosomal position effects, allowing reliable comparisons
of multiple transgenes individually integrated at the same genomic site [8]. Additionally,
independent receptor clones can be generated which are expected to be integrated in ran-
dom genomic locations, which offers the possibility of discarding or confirming the results
obtained for a given integration site. An alternative potential use of the RMCE assay is to
drive the integration of the recombination cassette to a particular and neutral (a deserted)
chromosomal location via homologous recombination; for example, on chicken DT40 cells
(see below) or embryonic stem (ES) cells. One of the more valuable advantages of this pro-
cedure is that, after the selection with gancyclovir, the exchange cassette does not need to
have a positive selectable marker, favoring a more reliable expression of the test transgene
and avoiding any undesired regulatory influence of the selectable gene frequently located
nearby. The RMCE protocol can also be used for the functional characterization of regulatory
elements or even for chromatin structure studies [31, 32]. When an optimal integration site is
defined, we can produce polypeptides in a controlled, sustained and reproducible manner for
use in different applications [33, 34]. DT40 technology is well established and widely used
by researchers.
