A USENCIA DE JUNTA CENTRAL DE USUARIOS

To exclude the influence of the genetic background on a given phenotype it is essential to use mouse inbred strains of high homozygosity (Silver, 1995). In this work, all experiments were performed using mice on a genetically pure C57 black 6 N (C57BL/6N) background. If not on a C57BL/6N background, mouse mutant- or transgenic lines were backcrossed for at

least 7 generations to 100 % pure C57BL/6N wt animals. Mice were housed in micro-isolation chambers in the mouse facility of the Institute of Genetics, Cologne. All experiments conducted had previously been described in the animal experimental form as such was confirmed by the local animal protection committee under reference number 93.2.10.31.07.186. All mice were humanely euthanized according to protocols set forth by the Landesamt für Natur, Umwelt und Verbraucherschutz NRW. In the following, mutant- and transgenic mouse strains used in this study are explained.

4.14.1.1 SMA-mice

The FVB.Cg-Tg(hSMN2)2Hung Smn1tm1Hung/J were purchased from The Jackson Laboratory (Bar Harbor, Maine (USA), Stock number: 005058) and served as SMA mouse model in this study. In these mice, Smn exon 7 is replaced by a targeted insertion of the hypoxanthine phosphoribosyl transferase (HPRT) cassette. Since the homozygous knockout of Smn (Smn^-/-) results in early embryonic lethality, transgenic mice with a 115-kb genomic DNA fragment encompassing the human SMN2 region from a human BAC library were generated (Hsieh-Li et al., 2000). Importantly, these mice carry 2 hSMN2 copies per integrate (Riessland et al., 2010). In a next step, the hSMN2 transgene was crossed into the existing SMA background. This way, normal living and fertile Smn^-/-;hSMN2^tg/tg animals could be generated. It is then possible to breed Smn^-/-;hSMN2^tg/tg mice with Smn^-/+ animals to produce 50 % SMA-offspring of the disease genotype Smn^-/-;hSMN2^tg/wt and 50 % of heterozygous control animals of the genotype Smn^+/-;hSMN2^tg/wt. In the course of this work this SMA mouse line is termed “Hung SMA” mouse line or the “Taiwanese mouse model”

referring to the fact that it was generated by a Taiwanese group (Hsieh-Li et al., 2000).

Originally, SMA-Hung mice were obtained on pure FVB genetic background by the Jackson Laboratory. In contrast to that, all used Cre-lines and PLS3V5-transgenic mice were bred onto a pure C57BL/6N background. To investigate possible disease modifying effects of PLS3V5 expression on the SMA background, SMA-Hung mice had to be backcrossed against C57BL/6N wt animals for 7 generations to reach 99 % genetic purity.

4.14.1.2 PLS3V5-transgenic mice

4.14.1.2.1 PLS3V5-floxed mice

In the scope of this work PLS3V5 transgenic mice were generated. An N-terminally V5-tagged version of human PLS3 (PLS3V5) was first cloned into a murine Rosa26 locus targeting construct (Kindly provided by Dr. Thomas Wunderlich, Cologne). Targeted transgenesis was performed using the Hybrid ES cell line V6.5 (Eggan et al., 2002). Since this cell line represents a mixture of 129/Sv and C57BL/6 genetic background the resulting PLS3V5-floxed line had to be backcrossed against C57BL/6N wt animals for 7 generations to reach 99 % genetic purity. In the targeting construct, the PLS3V5 coding sequence (cds) is

located downstream of the CMV enhancer/chicken β-actin (CAG) fusion promoter. However, a loxP sites flanked stop-cassette is present between promoter and PLS3V5 transgene preventing uncontrolled PLS3V5 expression. By breeding with tissue-specific Cre-strains conditional overexpression of the PLS3V5 transgene can be achieved. For a detailed description of the generation and nomenclature of the PLS3V5-floxed line see chapters 5.3.1 and 5.3.2.

4.14.1.2.2 PLS3V5-ubi mice

By intercrossing the PLS3V5-floxed line with the Cre-deleter line cytomegalovirus minimal promoter (CMV)-Cre (Schwenk et al., 1995), permanent deletion of the stop-cassette between promoter and PLS3V5-cds was achieved. In the resulting and from now on termed PLS3V5-ubi mice deletion of the stop-cassette resulted in ubiquitous expression of the transgene in nearly all tissues investigated (chapter 5.4.1.1). Neither animals heterozygous nor homozygous for PLS3V5 displayed any obvious outer phenotypes. PLS3V5-ubi animals are fully fertile and show a normal life expectancy. Since CMV-Cre mediated deletion includes germ cells, the deletion of the stop-cassette is inherited to further generations also in the absence of the CMV-Cre allele. For a detailed description of the generation and nomenclature of the PLS3V5-floxed line see chapters 5.3.1 and 5.3.3.

4.14.1.3 Cre-lines

4.14.1.3.1 CMV-Cre-deleter mice

The CMV-Cre deleter line expresses Cre-Recombinase under the control of the human cytomegalovirus minimal promoter (CMV) (Schwenk et al., 1995). Using this mouse line, deletion of loxP flanked genes or stop-cassettes occurs in all tissues, including germ cells.

For this reason and because it was available in the mouse facility of the Institute of Genetics (Cologne) the CMV-Cre line was chosen to permanently remove the stop-cassette between promoter and PLS3V5-cDNA in the PLS3V5-floxed line. In this context, it is important to mention that the CMV-Cre transgene is located on the X-chromosome (Schwenk et al., 1995). Due to gene silencing effects via X-chromosomal inactivation only CMV-Cre homozygous females (CMV-Cre^X_tg/X_tg) were used to efficiently delete the stop-cassette in F1 male mice of the genotype PLS3V5^fl_st/wt;CMV-Cre^X_tg/y (For a detailed explanation see chapter 5.3.3).

4.14.1.3.2 Hb9-Cre mice

In the Hb9-Cre mouse line the Cre gene is expressed under the intrinsic motor neuron-specific promoter of the Hb9 gene. Since homozygous loss of Hb9 results in perinatal lethality and because the Cre insertion disrupts Hb9 function (Arber et al., 1999, Yang et al., 2001) it is only possible to keep the Hb9-Cre transgene in a heterozygous state. By breeding

the PLS3V5-floxed line with Hb9-Cre mice, it was possible to delete the stop-cassette specifically in the motor neurons. This technique allows to investigate possible cell autonomous effects of PLS3V5 overexpression on motor neurons, nerve terminals as well as AChR clustering. The Hb9-Cre line was on pure C57BL/6N background.