Appendix - CNIC

SCIENTIFIC REPORT ´09 7 Technical Units
Viral vectors
> RESEARCH INTEREST
Head of Unit: Juan Carlos Ramirez
Research Scientist: Raúl Torres
Predoctoral Researcher: Zita Garate
Technician: Aida Garcia
The Viral Vectors facility develops efficient recombinant
viruses and strategies for gene transfer, using cloning
strategies based both on restriction enzymes and on
recombinases (cre, clonaseTM ). The viral stocks currently
produced, purified and titrated at the facility are second and
third generation lentiviruses; eco-, ampho- and xenotropic
pseudotyped retroviruses, and Ad5∆E1a–derived
adenoviruses. Our quality control procedures include
replication competent particle assays. In addition to cDNAencoding
gain-of-function vectors, we design vectors for lossof-function
studies encoding Pol II/Pol III-driven miRNAs or
shRNAs. Lentivectors designed to drive cre-mediated
expression of shRNAs for in vitro and in vivo studies are also
available. We are currently developing capacity for the
production of Ad-gutless and Adeno-associated viruses (AAV)
pseudotyped with 2, 8 and 9 capsids to enable targeted
delivery in vivo.
We also conduct our own technical and basic research
programs. Technical research projects include 1) the
development of novel tools based on non-integrative lentivirus
for efficient homologous recombination at specific loci in
mouse or human embryonic stem cells; 2) development of
strategies for the safe production of induced pluripotent stem
cells; and 3) the establishment of miRNA-based positive
sensor systems that will allow identification and selection of
subsets of native and induced stem cells with specific
pluripotencies and differentiation potentials. In collaboration
with the Comparative Medicine Unit (Experimental Surgery
Core) we are also implementing methods for efficient in vivo
delivery of lenti- and adeno-vectors to the heart.
Our basic research is currently focused on how the gene for
the chemokine SDF-1γ is specifically transcribed in heart cells
to drive nucleolar localization of the protein. To characterize
the role of this unprecedented feature and to define the
expression profile of cardiac SDF-1γ during development, we
are developing in vivo RNAi strategies using tetraploid
embryos and conditional knockout mice.
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The carboxy terminus of SDF-1γ contains a nucleolar
localization signal that targets the protein (fused to
cerulean fluorescent protein) to the nucleoli of
transfected human cells, labeling them blue.