Spastin targeting strategy and confirmation

RESULTS

66

RESULTS

A targeting vector with targeted exon 5 was obtained from KOMP also containing exons 6, 7 and 8 in the 3’ homology region (Figure 21b). The correct E. coli clone containing the targeting vector was selected from E. coli bacteria obtained from KOMP after the confirmation of the correct sequence by restriction digestion and sequencing (for a detailed description of the used KOMP targeting vectors see section 4.1.1 and Figure 18). Using the restriction endonuclease AsiSI, the selected targeting vector was quantitatively linearized and used for electroporation of ES cells derived from C57BL/6NJMA8 mice (Figure 21b and Figure 22).

– 10000 bp – 8000 bp – 6000 bp

Figure 22: Agarose gel confirming the linearization of the spastin targeting vector using the restriction endonuclease AsiSI. Lane1: Spastin targeting vector before restriction digestion; Lane 2: molecular weight marker (HyperLadder I, Bioline); Lane 3: Spastin targeting vector after AsiSI digestion.

4.2.1.1 Southern Blotting

To confirm the correct insertion of the targeting vector at the 5’ end of the homology arm, a probe for Southern Blotting (the binding sites are indicated in Figure 21a and c; for a detailed sequence information see chapter 7.5.3) was generated using the primers P20 and P21 (Table 8). After digestion using the restriction endonuclease EcoRV, the probe recognized a DNA-fragment of 18.5 kb in DNA extracts derived from wild type mice. Because the targeting vector contained an additional EcoRV restriction site, a 14.8 kb fragment could be observed in targeted spastin

KO1-st/WT ES cells and their progeny (see Figure 23a and b).

Figure 23: Confirmation of the correctly targeted murine spastin gene at the 5’ homology arm via Southern Blotting. EcoRV digestion of wild type DNA leads to an 18.5 kb fragment between exons 2 and 6 of the spastin gene. The correct insertion of the targeting cassette introduces a new EcoRV restriction site leading to a shorter 14.8 kb fragment detectable by the 5’ probe (see Figure 21 for strategy) in the KO-1st allele. a) The Southern Blot was performed using DNA extracts originating from ES-cell clone 1C11 (HET). b) The Southern Blot was done using DNA extracted from tail-tip biopsies from spastin^KO-1st-allele F2 generation littermates descending from ES-cell clone 1C11. WT: Spastin^WT/WT; KO: SpastinKO-1st/KO-1st; HET: Spastin^WT/KO-1st.

4.2.1.2 Long-range PCR

Because the generation of a 3’ probe for Southern Blotting did not lead to satisfactory results using DNA extracts derived from wild type mice (data not shown), a long-range PCR was established to confirm the correct insertion of the targeting vector at the 3’ end. As can be

RESULTS

68 concluded from Figure 21c, the forward primer P61 binds close to the third loxP site in the targeted locus, which originates from the targeting vector, whereas the reverse primer P23 (Table 8) binds downstream of the targeting vector region, which should be present both in wild type and in targeted alleles. As expected, a 5043 bp band could be observed in apastin^KO-1st -allele ES cells as well as in their progeny (Figure 24).

Figure 24: Agarose gel showing results from 3’ long-range PCR to confirm correct targeting of spastin^WT/KO-1st in animals (HET) derived from ES cell clone 1C11. Primers P61 and P23 were used to amplify a long-range PCR product of 5043 bp. M: molecular weight marker (HyperLadder I, Bioline).

Mating of KO-first-allele mice to a Flp-recombinase driver line leads to the excision of the LacZ- and the neomycin-phosphotransferase cassettes resulting in a floxed allele that is flanked by one FRT site and two loxP sites (Figure 21d). The usage of primers P41 and P89 in polymerase chain reaction can detect the presence of the additional loxP and FRT sites by a slight band shift of the PCR product from 1256 bp for wild type and 1515 bp for floxed mice. The lack of the wild type PCR product in homozygous floxed animals was used to additionally prove the insertion of the targeting vector at the locus of purpose (Figure 25).

Figure 25: Confirmation of correct targeting of the Spg4 gene in PCR using the primers P41 and P89. DNA extracts from spastin^WT/WT (+/+), spastin^WT/FL (+/FL) and spastin^FL/FL (FL/FL) animal tail-tip biopsies were used as template for the polymerase chain reaction.

4.2.1.3 Confirmation of spastin protein loss in brain lysates

To confirm the ultimate loss of spastin protein in spastin knockout mice, antibodies derived from mouse clone 6C6 (Table 4) were used. To confirm the specific binding of the antibodies to spastin, HEK293-TN cells were transfected with a pEGFP-spastin construct starting from methionine M85 (pEGFP-spastin-ΔM1; see Table 6). The proteins from the lysates were separated by SDS-PAGE and analyzed by Western Blotting using the 6C6 spastin and anti-GFP antibodies (Figure 26).

In pEGFP-transfected HEK cells, a 30 kDa fragment was detected using the anti-GFP antibody and a band of approximately 50 kDa when using the spastin 6C6 antibody (lanes 1 and 5). In non-transfected control cells (lanes 2 and 6), no band could be observed using the GFP antibody and a band at the same molecular weight as in pEGFP-transfected-cells when using the anti-spastin antibody. Contrarily, in pEGFP-anti-spastin-ΔM1-transfected cells, two fragments of 75 kDa

RESULTS

and approximately 50 kDa were detected for both the GFP and the spastin antibodies (lanes 3 and 7 respectively). The 75 kDa band approximately corresponds to the expected molecular weight of the GFP-ΔM1-spastin fusion protein. The 50 kDa band has approximately the molecular weight expected for spastin starting from methionine M85/87.

kDa:

100 – 75 –

– – 25 –

Figure 26: Western Blotting of pEGFP-transfected, untransfected, and pEGFP-Spastin-ΔM1-transfected HEK-cell lysates using the mouse monoclonal 6C6 anti-spastin antibody or the rabbit anti-GFP antibody (SIGMA). GFP: pEGFP-transfected cells, C: untransfected control cells, GFP-Spast: pEGFP-Spastin-ΔM1-transfected cells.

The latter antibody was subsequently used to determine the loss of spastin expression in Western Blotting using full-brain lysates derived from spastin KO 1-st/KO 1-st and spastin^KO/KO mutant mice separated by SDS-PAGE. As can be seen in Figure 27a and b, the bands at the expected approximal molecular weights of spastin (approximately 50 and 60 kDa corresponding to the isoforms starting from methionine M85) can no longer be detected both in spastin KO 1-st/KO 1-st and spastin^KO/KO mutants. The quantification of signal intensities from heterozygous spastin^{KO 1-st/WT} animals revealed a reduction of approximately 54 % (Figure 27a and c). A one-way ANOVA revealed that the means of the measured spastin signal intensities differed significantly for the three genotypes F (2, 15) = 46.85, p<0.0001. Sidak’s multiple comparisons test yielded that spastin levels were significantly reduced in both heterozygous mice (t=5.478, df=15, p=0.0001) as well as in knockout mice compared to wild types (t=9.650, df=15, p<0.0001).

a) b) c)

Figure 27: Reduced spastin protein levels in prenatal spastin KO mice as revealed by Western Blotting using the 6C6 anti-spastin antibody on whole brain lysates. a) Western Blotting analysis of lysates from spastin^WT/WT (+/+), spastin^WT/KO-1st (+/d) and spastinKO-1st/KO-1st (d/d) littermates. A primary antibody directed against actin was used as a loading control. b) Western Blotting analysis of lysates derived from null-allele mutant spastin^WT/WT (+/+) and spastin^KO/KO (-/-) littermates. An antibody directed against GAPDH was used as a loading control. c) Quantification of spastin protein loss in prenatal spastin KO mice after normalization to the mean signal intensity of each tested group. The error bars represent the SEM.

wt het ko

0 50 100 150 200 250

genotype for spastin

signal intensity (AU)

upper band spastin

RESULTS

70 The latter antibody was also used to determine the reduction of spastin protein levels in forebrain-specific spastin KO animals (spastin^FL/FL/ CamKIIα-Cre^WT/TG). Brains from 15-week-old mice were isolated and separated into olfactory bulb, cortex, hippocampus, midbrain and cerebellum. After SDS-PAGE of the brain lysates, the 6C6 anti-spastin antibody was used for Western Blotting analysis. The spastin-specific signal intensities were normalized to the ones for PAN-cadherin or for NSE, which were used as loading controls. In contrast to mice with full spastin knockout, conditional spastin KO animals only showed a reduction of signal intensity for the upper of the two bands detected by the anti-spastin antibody. Interestingly, there was no difference in the lower band intensities between spastin^WT/WT/CamKIIα-Cre^WT/TG and spastin^FL/FL/CamKIIα-Cre^WT/TG littermates (Figure 28a).

kDa:

75 –

150 – 100 – 50 – 37 –

Spastin

PAN-Cadherin NSE

Figure 28: Conditional targeting of spastin. a) Western Blotting analysis of spastin expression in brain lysates obtained from 15 weeks spastin^WT/WT/CamKIIα-Cre^WT/TG (termed WT) and spastin^FL/FL/ ^/CamKII- cre^WT/^TG littermates (termed KO). The mouse anti-spastin antibody (clone 6C6) was used. PAN-cadherin and NSE were used as loading controls. OB: olfactory bulb; CX: cortex; HC: hippocampus; MID: midbrain; CB: cerebellum.

The Western Blotting upper band signal intensities using the 6C6 anti-spastin antibody revealed a mean reduction by 83 % for the cortex, 69 % for the hippocampus and by 80 % for the midbrain in adult spastin^FL/FL/ CamKIIα-Cre^WT/TG mice. The results could not be tested for significance due to a small sample size.

4.2.1.4 Confirmation of spastin protein loss in immunocytochemistry

Neurons were cultivated from hippocampi isolated from spastin^WT/WT animals and spastin^KO/KO littermates at postnatal day 0 to confirm spastin protein loss in the KOs. As can be seen in Figure 29a, the usage of the 6C6 primary antibodies followed by Alexa 488 anti-mouse secondary antibodies led to reduced fluorescence signal intensities as detected by confocal laser-scanning microscopy. A significant difference in the mean fluorescence intensities between the genotypes obtained from 4 different experiments could be confirmed with an ordinary one-way ANOVA:

F(2, 76)=79.13, p<0.0001. A post hoc Tukey test showed that the means between WT vs. HET, WT vs. KO and HET vs. KO differed significantly (p<0.0001, <0.0001 and p=0.0001, respectively).

OB CX HC MID CB 0

5 10 15 20 25

brain region

spastin expression (AU)

WT KO

!!!!!!!OB!!!!!!!!!!!!!!!!!CX!!!!!!!!!!!!!!!!!!!!!HC!!!!!!!!!!!!!!!!!MID!!!!!!!!!!!!!!!!!CB WT!!!!!KO!!!!!!WT!!!!!!KO!!!!!!!WT!!!!!!KO!!!!!!!!WT!!!!!!KO!!!!!!!WT!!!!!KO S

pC

a) !!!!!!!!!!!!!!!b)

RESULTS

The mean fluorescence intensities were reduced by 41 % for HET and by 74 % for KO when compared to the mean fluorescence intensities from WT pointing to an intermediate reduction of spastin protein levels for heterozygous animals.

a) WT spastin KO b)

phalloidin spastin

spastin only

Figure 29: Immunocytochemistry using the spastin 6C6 antibody. A) Exemplary picture showing cultivated postnatal day 0 primary hippocampal neurons derived from spastin^WT/WT (termed WT) animals and spastin^KO/KO (termed KO) littermates that were stained using the monoclonal 6C6 anti-spastin antibody (green) and phalloidin (red). B) Quantification of the mean fluorescence intensities obtained from four independent experiments (with n=35 measurements for WT, n=15 for HET, and n=29 for KO). The measured fluorescence intensities were normalized to the mean fluorescence intensity from WT. Scale Bar: 20 µm