The aim of the project is twofold

The aim of the project is twofold. The first part of the project is concerned with global activation of the N598R point mutation in the N R l subunit of the NMDA receptor and is of analytical nature, whereas the second part is concerned with regionally restricted activation and consists primarily in genetic engineering.

1.6.1 Global activation

Global activation of the NRl N598R mutation is achieved by mating NRl^®®^"^ mice with the Cre ‘Deleter’-mice, which carry an X-chromosomally linked Cre-transgene and are expressing Cre-recombinase globally (Schwenk et al., 1995). We have found that global activation of the N R l N598R mutation is dominant negative lethal. By employing the N Rl knockout allele in addition, it is possible to generate mice with five different N R l genotypes. I have investigated the effect of altered NMDA receptor function in the developing somatosensory system of the mouse, which is a suitable model for the study of developmental synaptic plasticity. I find that the N598R mutation in the N R l subunit affects the formation of whisker-related patterns (barrelettes) in the brainstem of newborn pups.

1.6.2 Regionally restricted activation

The aim of this line of the project is to generate a mouse model with restricted expression of Cre in order to become able to investigate the effect of altered NMDA receptor function in subregions of the brain. The dominant negative effect of the N R l N598R mutation implies that the mutation must not be activated in regions of the brain that are essential for the viability of the mice.

1.6.2.1 Gene targeting strategy

I attempt to couple the expression of Cre, fused with EGFP (EGFP-Cre), to the expression of the endogenous gene for the kainate receptor subunit K A l (Figure 1-8). The K A l gene was chosen, because it is expressed to a very limited extent in

the brain. More specifically, it is predominantly expressed in CA3 pyramidal cells of the hippocampus, and to some extent in dentate gyrus granule cells (W erner et al., 1991), regions of interest for the study of synaptic plasticity as well as learning and memory. In the targeted locus, the EGFP-Cre coding sequence is inserted into the 3 ’ untranslated region (3’UTR) of the K A l gene, fusing its transcription with the one of KAl . The resulting mRNA is bicistronic. Translation of EG FP-Cre is initiated at an internal ribosome entry site (1RES) which allows initiation of translation independent from a 5 '-cap structure (Mountford et ah, 1994; M ountford and Smith, 1995).

A

ribosome

V

KA1-mRNA ^

STOP

3’UTR

B

i

ribosome

STOP

t

KA1/Cre

-mRNA

(

T)

i

Figure 1 -8 T he g e n e targeting strategy. (A) Translation of th e wildtype KAI-mRNA is initiated at the 5 ’-cap and term in ates at th e sto p c o d o n . (B) Translation of a modified, bicistronic KA1/ E G FP-C re mRNA. Translation o f E G FP -C re is initiated by an Internal R ib o s o m e Entry Site (1RES). The targeting e l e m e n t s are inserted into the 3' untranslated region (3’UTR) of the e n d o g e n o u s KA1-gene using h o m o lo g o u s recombination in ES-cells.

A relatively new technical aspect of the strategy is that the targeting vector for modification of the K A l-gene in ES-cells is assem bled using a com bination of conventional cloning in bacteria and cloning by homologous recom bination in yeast (Figure 1-9). Therefore, the targeting vector contains genes for selection in ES-cells and in yeast. In addition, the selection markers are flanked by frt-sites and hence can

be rem oved using the Flp/frt recom bination system (Dymecki, 1996; Rodriguez et al., 2000).

Once the selection cassette is removed by Flp-recom binase, the control of the expression of the engineered K A l-C re fusion transcript should be the same as of the wild-type K A l gene, i.e. expression should be restricted to K A l-expressing tissues. After analysis of the expression patterns of Cre and K A l, this mouse will eventually be crossed with the mouse to activate the dom inant negative mutation in a K Al-tissue-specific manner. S T O P Targeting site K A l- g e n e on YAC polyA-signal — U — — # his3 1RES EGFR-C n e o frt frt B STO P polyA-signal his3 1RES n e o c e n f1 (+)ori Ieu2

Figure 1 -9 Strategy for cloning of the ES-cell targeting vector. (A) Insertion of the targeting c a s s e t t e into the KA1-3’UTR in a KA1-YAC: In E .coli the targeting e l e m e n t s are cloned - internal ribosom e entry site (1RES) followed by an EG FP-C re translation unit, followed by se lection markers neo, for positive se lection in ES-cells, and his3, for se lec tion in yeast. The se lection markers are flanked by frt-sites (frt, Flp recognition target) and can b e removed by F lp -recom b in ase. A targeting c a s s e t t e is isolated from the cloning vec to r a s a c a s s e t t e consis tin g of the targeting e le m e n ts flanked by K A1-hom ology arm s for integration into the K A l - 3 ’UTR by d o u b le c r o s s o v e r in y e a s t . (B) T h e final E S -cell targeting construct is obtained from the YAC a s a shuttle vector using a y e a s t e p iso m a l vector. This vector also carries the HSV thymidine kinase g e n e (HSVtk) for n egative selection in ES-cells.