cDNA and Peptide Libraries
power of complementation cloning, long appreciated in bacterial and
yeast genetic systems, may now be more fully accessed for mammalian
cells by applying retroviral delivery systems.
basic tenet of the approach is that using retroviruses large libraries
of encoded objects (cDNAs, peptides, RNAs)-- among them desired objects
capable of exerting a dominant phenotype on target cells-- can be transferred
to target mammalian cells. By applying appropriate genetic selections
one can isolate those cells expressing the object of interest from among
a background of unwanted objects.
have applied this in two main areas:
libraries. In this case a cDNA library is made in a retroviral
backbone from a source cell type. The cDNA library (generally around
5 x 10^6 independent full-length inserts) is converted into packaged
retrovirus and used to infect a target cell. After infection of
the target cell a suitable genetic selection is set that allows
for one to distinguish those cDNAs capable of conferring a new phenotype
on the target cell. We have used this approach to isolate cDNAs
that block a variety of apoptotic stimuli as well as several surface
markers. Published papers on this work can be found in Kitamura
et al and Hitoshi et al, among others in the Publications section.
A library of short peptides, termed DOMINANT EFFECTORS, are encoded
into the retroviral backbone. The peptides can be considered "shapes"
in the pharmaceutical sense of the word (see the mini-tutorial
link at the right for more info). An expectation is that rare
peptides will be capable of interfering with intracellular signaling
systems in interesting and novel ways. A further prediction is that
the selected peptides will be useful for identifying the signaling
molecules upon which they act (target identification). This can
lead to reagents that can be used to understand function of biological
pathways and discern new drug candidates. A publication of our work
in this area can be found in the following Nature Genetics article
et al "Dominant Effector Genetics in Mammalian Cells".
A News and Niews on the work can be found at this link. We have
also used this approach to create novel inhibitors of T cell pathways
and HIV-1 replication (Shigemi Kinoshita and GPN, unpublished).
Background articles can be also found in Lorens
et al and
basic library principles in the cDNA
publication of Kitamura
T, et al Efficient screening of retroviral cDNA expression
of libraries include:
cDNA libraries in which PCR or other mutagenesis
is used to mutate a known cDNA and a library selection system is set to
find those mutant cDNAs that survive the selection. This can be used to
find, in a non-biased and interesting manner, new sites on proteins that
are not readily found using standard mutagenic approaches.
libraries. Pioneered by Igor Roninson at the University of Chicago
this approach uses randomly fragmented cDNA expressed in retroviral backbones.
The approach allows for "mini-domain" interference in signaling
systems with the notion that the fragments' ability to interfere, and
its constitution, allows for biological inferences to be made.
Libraries. A variety of ribozymes have been encoded in retroviruses.
Some individuals have suggested that by randomizing the targeting sequence,
and setting appropriate selection criteria, one can isolate ribozymes
with specific properties that are capable of cleaving "unknown"
mRNAs. Of course, the inference is that once the ribozyme is sequenced
one will be able to soon discover the nature of the target RNA and then
determine how the cleavage event determines the selected event.
of interest is the PNAS manuscript on which this cDNA library work was
based by Kitamura
T, et al.
Genetics News and Views on the Xu et al manuscript.