Rapid construction in yeast of complex targeting vectors for gene manipulation
in the mouse
Rapid construction in yeast of complex targeting vectors for gene manipulation in the mouse
Thorsten
Storck*
,
Ulrich
Krüth
,
Rohini
Kolhekar
,
Rolf
Sprengel
and
Peter H.
Seeburg
Center for Molecular Biology (ZMBH), University of Heidelberg, INF 282 and Max-Planck Institute for Medical Research, Jahnstrasse 29, 69120
Heidelberg
,
Germany
Received July 21, 1996
;
Revised and Accepted October 7, 1996
DDBJ/EMBL/GenBank accession nos U63018, U63120
ABSTRACT
Targeting vectors for embryonic stem (ES) cells typically contain a mouse gene
segment of >7 kb with the neo gene inserted for positive selection of the
targeting event. More complex targeting vectors carry additional genetic
elements (e.g. lacZ, loxP, point mutations). Here we use homologous
recombination in yeast to construct targeting vectors for the incorporation of
genetic elements (GEs) into mouse genes. The precise insertion of GEs into any
position of a mouse gene segment cloned in an
Escherichia coli
/yeast shuttle vector is directed by short recombinogenic arms (RAs) flanking
the GEs. In this way, complex targeting vectors can be engineered with
considerable ease and speed, obviating extensive gene mapping in search for
suitable restriction sites.
Our approach utilizes the high recombination efficiency in yeast which permits
the insertion of genetic markers at precise chromosomal positions by gene
replacement (
1
,
2
). Typically, yeast cells are transformed with a replication incompetent DNA
fragment containing a marker gene flanked by sequences homologous to the
integration site, which promote two homologous recombination events. To adopt
this strategy for our purposes, a mouse gene segment of >7 kb which provides
the backbone of a targeting vector for ES cells, isolated from a [lambda] genomic library, is cloned in an
Escherichia coli
/yeast shuttle vector. In parallel, all GEs to be inserted into the gene
segment, including the neo gene and the yeast selection marker URA3, are
assembled by cloning into a suitable bacterial plasmid (e.g. the pRAY vectors
described below and in Fig.
1
). The GEs are then collectively flanked by short (200-400 bp) sequences, PCR-derived from a location in the mouse gene segment where GE
insertion is desired. These short sequences which direct GE insertion are
termed recombinogenic arms (RAs). Finally, to transfer the GEs into the gene
segment on the shuttle vector, the entire GE cassette flanked by RAs is
released from the bacterial vector and transfected as donor DNA into yeast
cells together with the recombinant shuttle vector as the recipient (Fig.
1
). Homologous recombinants are selected by URA and plasmids from selected
colonies are then transferred to
E.coli
. Transfer to
E.coli
is necessary because not all copies of the recombinant shuttle vector in a yeast
cell undergo homologous recombination. It also facilitates plasmid growth for
further DNA analysis.
