Download transgenic mice core unit

Direction of Innovation
TRANSGENIC MICE
CORE UNIT
Biotechnology Programme | Transgenic Mice Core Unit
Sagrario Ortega
Core Unit Head
Graduate Student
Aleida Pujol
Technicians
Estefania Ayala ( since February ),
M. Carmen Gómez, Jaime A. Muñoz
( TS )*, Lucía M. Pérez De Ayala ( since
December ) ( PEJ-L )**, Patricia Prieto
( TS )*, Pierfrancesco Vargiu ( TS )*
*Titulado Superior ( Advanced Degree )
**Plan de Empleo Joven-Licenciado ( Youth
Employment Plan-Graduate )
RESEARCH HIGHLIGHTS
During 2015, the Transgenic Mice Unit has focused on the
establishment of 2 new technologies : the use of mouse haploid
embryonic stem cells ( hESCs ) for cancer-related genetic
screenings, and the incorporation of the CRISPR/Cas9 system
for genome editing in mice.
The first mouse hESC lines were established in 2011, by Anton
Wutz and Joseph Penninger, from wild type parthenogenetic
embryos. Since then, they have been shown to be a powerful
tool for genomewide forward- and reverse- genetic screenings
in mice using transposons or gene-trap lentiviruses for
genomewide mutagenesis. The haploid karyotype enables
direct phenotypic selection of recessive mutations that would
be silent in a diploid context. We are interested in exploiting
the potential of mouse hESCs for cancer related screenings
by generating parthenogenetic hESCs from cancer mouse
models and mutant mice available at the CNIO. For this
purpose we are creating a collection of mutant hESCs, called
HaploESCancer collection, derived from CNIO mice. This
year we have established haploid mouse embryonic stem
cell lines from p53 KO, Brca1_lox and ATR_lox mice by SrCl2
activation of mouse oocytes. These cells will be mutagenised
using PiggyBac transposition − ENU or CRISPR/Cas9 − in
order to obtain mutant clones covering nearly the whole set
of protein/RNA coding loci in the mouse genome.
OVERVIEW
Genetically modified mice are an essential tool for analysing the
molecular mechanisms underlying tumour development and
cancer biology. Modelling cancer by modifying the germ line of the
mouse has become a crucial component of drug discovery as well
as for the assessment of experimental therapies at the preclinical
stage. The Transgenic Mice Unit at the CNIO offers state-of-theart technology for the manipulation of the mouse genome. Using
classical transgenesis, homologous recombination in embryonic
stem cells and genome editing by targeted nucleases, the Unit
has generated more than 300 mutant alleles of cancer related
genes in the mouse germ line. The Unit also provides support
and collaborates with CNIO researchers in many aspects related
to research with embryonic stem ( ES ) cells, induced pluripotent
stem ( iPS ) cells, and embryo- and mouse model-based research.
Finally, the Unit leads its own research projects focused on the
ANNUAL REPORT 2015
“ In 2015, the Unit has contributed
to 6 peer review articles, in
collaboration with CNIO and
external groups, including projects
related to the characterisation of
cell cycle regulator functions in vivo
and in tumour development, as well
as of new mechanisms of lymphatic
system development.”
The CRISPR/Cas9 system, imported from bacterial and archaeal
genomes, has expanded the currently available set of mammalian
genome engineering tools, providing an easy, efficient, flexible
and versatile method of introducing targeted mutations in
mammalian genes. The CRISPR/Cas9 system has also been used
to generate knockout and knockin mice by introducing the guide
CRISPR RNA and the Cas9 RNA directly into mouse zygotes.
We have succesfully used this system directly in mouse zygotes
for generating deletions of regulatory regions and knockout
alleles by open reading frame alteration. We are now interested
in exploring the advantages of this system for precise genome
editing with respect to gene targeting in mouse embryonic stem
cells ( FIGURE ). s
Figure Use of the CRISPR/Cas9
system for genome editing in the
mouse. The figure illustrates the
different strategies and delivery
∞∞
∞∞
∞∞
∞∞
∞∞
mechanisms used to target any given
sequence of genomic DNA in the germ
line of the mouse using zygotes or
mouse embryonic stem cells ( ES cells ).
PUBLICATIONS
Martinez-Corral I, Ulvmar MH, Stanczuk
L, Tatin F, Kizhatil K, John SW, Alitalo K,
Ortega S, Makinen T ( 2015 ). Nonvenous
origin of dermal lymphatic vasculature.
Circ Res 116, 1649-1654.
Trakala M, Rodríguez-Acebes S, Maroto
M, Symonds CE, Santamaría D, Ortega
S, Barbacid M, Méndez J, Malumbres
M. ( 2015 ). Functional reprogramming
of polyploidization in megakaryocytes.
Dev Cell 32, 155-167.
Stanczuk L, Martinez-Corral I, Ulvmar MH,
Zhang Y, Laviña B, Fruttiger M, Adams
RH, Saur D, Betsholtz C, Ortega S, Alitalo
K, Graupera M, Mäkinen T ( 2015 ). cKit
Lineage Hemogenic Endothelium-Derived
Cells Contribute to Mesenteric Lymphatic
Vessels. Cell Rep. PMID : 25772358.
López-Iglesias P, Alcaina Y, Tapia N, Sabour D, Arauzo-Bravo MJ, Sainz de la Maza
∞∞
∞∞
D, Berra E, O’Mara AN, Nistal M, Ortega S,
Donovan PJ, Schöler HR, De Miguel MP.
( 2015 ). Hypoxia induces pluripotency in
primordial germ cells by HIF1a stabilization
and Oct4 deregulation. Antioxid Redox
Sign 22, 205-223.
Viera A, Alsheimer M, Gómez R, Berenguer
I, Ortega S, Symonds CE, Santamaría D,
Benavente R, Suja JA ( 2015 ). CDK2 regulates nuclear envelope protein dynamics and telomere attachment in mouse
meiotic prophase. J Cell Sci 128, 88-99.
Búa S, Sotiropoulou P, Sgarlata C, Borlado, LR, Eguren M, Domínguez O, Ortega
S, Malumbres M, Blanpain C, Méndez J
( 2015 ). Deregulated expression of Cdc6
in the skin facilitates papilloma formation
and affects the hair growth cycle. Cell
Cycle 14, 3897-3907.
generation of mouse models to study tumour biology, as well as
on the screening of cancer-related genes.
148
SPANISH NATIONAL CANCER RESEARCH CENTRE, CNIO
149