Downregulation of genes in TG-13 compared to WT

Accession No. Gene Name Product

gb:BC027434.1 hemoglobin, beta adult minor chain gb:NM_013732.1 Cart-pending cocaine and amphetamine regulated transcript gb:BC024599.1 Unknown (protein for MGC:30545) gb:AK011116.1 Hba-a1

gb:BB027654 5730450N06Rik RIKEN cDNA 5730450N06 gene gb:NM_009052.1 Rex3 reduced expression 3

gb:AK011116.1 Hba-a1

Table 4. DNA and RNA expression of clonal cells

+ : DNA/RNA expression level; – :no DNA/RNA expression; L:

leaky expression

c1 c2 c3 c4 c5 c6 c7 c8 c9

DNA + — ++ + — + + ++ ++

RNA — — + — — — +(L) + +(L)

c10 c11 c12 c13 DNA ++ ++ ++ ++

RNA +(L) +(L) +(L) +(L)

Fig. 1. construct for generation of B1 overexperssion transgenic mice.

We use the NSE promoter to replace the CMV promoter in

Bβ1-pFLAG-pEGFPN1 (Kindly provided by Dr. Strack). First, CMV was cut out by AseI and NheI and both ends were blunt-ended. NSE was

released from pEGMT-pNSE by SphI and NseI digested and blunt-ended.

Both the pCMV-deleted vector and NSE promoter fragments were ligated and transformed into E.coli host to obtain the expected transgenic

constructs.

Fig. 2. Rotarod behavior of mice at early stages.

The rotarod speed was accelerated from 4 to 30 rpm in the first 300 sec, and then maintained at 30 rpm till maximum duration, 600 sec. There were no significant difference in line TG-13 (A) and line TG-20 (B) transgenic mice compared to wild-type mice (mean±S.E.M.).

Testing Day

Latencies (second)

0 100 200 300 400 500 600

TG 13 (n=12) control (n=10)

28w 32w 36w 40w 44w 48w 60w

Testing Day

Latencies (second)

0 100 200 300 400 500 600

TG 20 (n=7) control (n=6)

26w 30w 34w 38w 42w 46w 58w

Fig. 3. Rotarod behavior of mice at later stages.

The rotarod speed was accelerated from 2 to 20 rpm in first 300 sec, and then maintained at 20 rpm till maximum duration, 600 sec. There were significant difference in line TG-13 (A) and line TG-20 (B) transgenic mice compared to wild-type mice (mean±S.E.M.).*, p<0.05

Age (months)

Latencies (seconds)

0 100 200 300 400 500

600 WT (n=10) TG13 (n=9)

16 17 18

* * *

Age (months)

Latencies (seconds)

0 100 200 300 400 500

600 WT (n=6)

TG20 (n=7)

16 18

* * * *

A. Line TG-20 compared to wild type mice.

Fig. 4. Changes in gene expression between heterozygous transgenic and wild type mice.

(A) Line TG-20 compared to wild type mice (B) line TG-13 compared to wild type mice (C) line TG-20 compared to line TG-13.

B. Line TG-13 compared to wild type mice.

C. Line TG-20 compared to line TG-13.

Fig. 5. IHC results of 3-month-old mouse cerebellum.

(A) calbindin staining (B) NeuN staining(C) quantitation of calbindin staining (D) quantitation of NeuN staining (scale bar: 50 µm)

(mean±S.E.M.).* p < 0.05

Fig. 6. IHC results of 3-month-old mouse cerebellum.

(A) GFAP staining (B) flag staining (C) quantitation of GFAP staining (D) quantitation of flag staining (scale bar: 50µm) (mean±S.E.M.).* p < 0.05

WT TG20 TG13

GFAP

Relative expression level

0.0 0.5 1.0 1.5 2.0

2.5 WT (n=3)

TG20 (n=3) TG13 (n=3)

flag

Relative expression level

0.0 0.5 1.0 1.5 2.0

2.5 WT (n=3)

TG20 (n=3) TG13 (n=3)

Fig. 7. IHC results of 3-month-old mouse cerebellum.

(A) CART staining (B) GH staining (C) quantitation of CART staining (D) quantitation of GH staining (scale bar: 50µm) (mean±S.E.M.).* p < 0.05

WT TG20 TG13

Fig. 8. IHC results of 7-month-old mouse cerebellum.

(A) calbindin staining (B) NeuN staining (C) quantitation of calbindin staining (D) quantitation of NeuN staining (scale bar: 50µm)

(mean±S.E.M.).* p < 0.05

Fig. 9. IHC results of 7-month-old mouse cerebellum.

(A) GFAP staining (B) flag staining (C) quantitation of GFAP staining (D) quantitation of flag staining (scale bar: 50µm) (mean±S.E.M.).* p < 0.05

WT TG20 TG13

Fig. 10. IHC results of 7-month-old mouse cerebellum.

(A) CART staining (B) GH staining (C) quantitation of CART staining (D) quantitation of GH staining (scale bar: 50µm) (mean±S.E.M.).* p < 0.05

WT TG20 TG13

Relative expression level

0.0 0.5 1.0 1.5

2.0 WT (n=3)

TG13 (n=3) TG20 (n=3)

CART

C *

Relative expression level

0.0 0.5 1.0 1.5 2.0

2.5 WT (n=3)

TG13 (n=3) TG20 (n=3)

Fig. 11. IHC results of 19-month-old mouse cerebellum.

(A) calbindin staining (B) NeuN staining (C) quantitation of calbindin staining (D) quantitation of NeuN staining (scale bar: 50µm)

(mean±S.E.M.).* p<0.05

Fig. 12. IHC results of 19-month-old mouse cerebellum.

(A) GFAP staining (B) flag staining (C) quantitation of GFAP staining (D) quantitation of flag staining (scale bar: 50µm) (mean±S.E.M.).* p<0.05

WT TG20 TG13

20WT 20TG 13TG

GFAP

Relative expression level

0.0 0.2 0.4 0.6 0.8 1.0 1.2

1.4 20WT (n=3)

TG20 (n=3) TG13 (n=3)

flag

Relative expression level

0.0 0.5 1.0 1.5 2.0 2.5

3.0 20WT (n=3)

TG20 (n=3) TG13 (n=3)

C D

Fig. 14. IHC results of 19-month-old mouse cerebellum.

(A) CART staining (B) GH staining (scale bar: 50µm)

Due to this IHC analysis only conducted on 2 mice (n=2), thus no quantitative data shown in this figure.

WT TG20 TG13

20WT 20TG 13TG

Fig. 14. Western blot analysis of 2-month-old mouse brain and cerebellum.

(A) western blot analysis using GFAP (51 kDa), NeuN (three isoforms were 66, 48 and 46 kDa) and calbindin (28 kDa) antibodies staining, indicate that decreased neuron number in transgenic mice, but calbindin and GFAP were no changed, B :brain, C :cerebellum (B)(C)(D)

quantitative analysis data (protein loading 50µg) (mean±S.E.M.).* p<0.05

B C B C B C

Fig. 15. Western blot analysis of 7-month-old mouse brain and cerebellum.

(A) western blot analysis showed decreased neuron cell and PC number in transgenic mice, but GFAP was increased (B)(C)(D) quantitative analysis data (protein loading 50µg) (mean±S.E.M.).* p<0.05

B C B C B C

Fig. 16. Western blot analysis of 19-month-old mouse brain and cerebellum.

(A) western blot analysis showed GFAP was increased and PC loss in transgenic mice, but neuron cell were no changed (B)(C)(D) quantitative analysis data (protein loading 50µg) (mean±S.E.M.).* p<0.05

Relative expression level

Fig. 17. Western blot analysis of CART staining of heterozygous transgenic mice brain and cerebellum.

(A) western blot analysis using CART (14 kDa) antibody staining, CART signal were somehow decreased in transgenic mice. Quantitative analysis data of (B) 2-month-old (C) 7-month-old (D) 19-month-old mice (protein loading 50µg) (mean±S.E.M.).* p<0.05

B C B C B C

Fig. 18. Western blot analysis of tau phosphorylation of heterozygous transgenic mice brain and cerebellum.

(A) western blot analysis using tau antibody staining, p-tau (55 kDa) and tau (44 kDa), there were no change about p-tau/tau level. Quantitative analysis data of (B) 2-month-old (C) 7-month-old (D) 19-month-old mice (protein loading 50µg) (mean±S.E.M.).* p<0.05

WT TG20 TG13

brain c erebellum brain cerebellum

p-tau tau

19M p-tau/tau

Fig. 19. Western blot analysis of PP2A activity of heterozygous transgenic mice brain and cerebellum.

(A) western blot analysis using PP2A-C (36 kDa) antibody staining, the of PP2A was only elevated in cerebellum of 7-month-old mice.

Quantitative analysis data of (B) 2-month-old (C) 7-month-old (D) 19-month-old mice (protein loading 50µg) (mean±S.E.M.).* p<0.05 19M

Fig. 20. Result of Rotarod performance of homozygous line TG-13.

An representative data of semi-quantitative PCR for homozygous

transgenic mice identification (* homozygous) (B) result of rotatod test, the performance of wild type mice is significantly better than

homozygous line TG-13 mice at difference stages (C) mouse body weight at difference stages (mean±S.E.M.).*, p < 0.05

M 1* 2 3* 4 5 6 7 8* N

3rd 4th 1st 2nd 1st 2nd 1st 2nd

2 3 4 5 (months)

Fig. 21. Result of Rotarod performance of homozygous line TG-20.

(A) result of rotatod test, the performance of wild type mice is

significantly worse than homozygous line TG-13 mice at 2 month old (B) mouse body weight at 2-month-old (mean±S.E.M.).* p<0.05

Weight (g)

Fig. 22. Result of open-field locomotor activity test.

8-week-old mice were put into a 30 cm* 30 cm* 30 cm white box and detected by Etho-Vision video tracking system. Analysis of (A) distance moved and (B) rearing frequency are shown. (mean±S.E.M.).* p<0.05

Distance moved (cm)

0 1000 2000 3000

4000 WT (n=20) TG20 (n=9) TG13 (n=9)

Rearing Frequency

0 5 10 15 20

25 WT (n=20)

TG20 (n=9) TG13 (n=9)

* *

Fig. 23. IHC results of 3-month-old mouse cerebellum.

(A) calbindin staining (B) NeuN staining (C) GFAP staining (scale bar:

50µm)

WT TG20 TG13

B A

WT TG20 TG13

Fig. 24. IHC results of 3-month-old mouse cerebellum.

(A) flag staining (B) CART staining (C) GH staining (scale bar: 50µm)

WT TG20 TG13

Fig. 25. IHC results of 6-month-old mouse cerebellum.

(A) calbindin staining (B) NeuN staining (C) GFAP staining (scale bar: 50µm)

WT TG20 TG13

Fig. 26. IHC results of 6-month-old mouse cerebellum.

(A) flag staining (B) CART staining (C) GH staining (scale bar: 50µm)

WT TG20 TG13

Fig. 27. IHC results of 14-month-old mouse cerebellum.

(A) calbindin staining (B) NeuN staining (C) GFAP staining (scale bar: 50µm)

WT TG20 TG13

C B

Fig. 28. IHC results of 14-month-old mouse cerebellum.

(A) flag staining (B) CART staining (C) GH staining (scale bar: 50µm)

WT TG20 TG13

Fig. 29. Western blot analysis of 4-month-old homozygous mouse brain and cerebellum.

(A) western blot analysis showed GFAP was slight increased and neuron cell loss in transgenic mice, but purkinje cell were no changed (B)(C)(D) quantitative analysis data (protein loading 50µg) (mean±S.E.M.).* p<0.05

B C B C B C

Fig. 30. Western blot analysis of 4-month-old homozygous mouse brain and cerebellum.

(A) western blot analysis showed there were no difference of p-tau/tau level, but PP2A C subunit has increased in homozygous transgenic mice cerebellum, CART was no chahged (B)(C)(D) quantitative analysis data (protein loading 50µg) (mean±S.E.M.).* p<0.05

B C B C B C

Fig. 31. Behavior detected by HomeCageScan

(A) rest (B) awaken (C)

twitch (D) hang (E) stretch (F) jump (G) distance traveled (mean±S.E.M.).* p<0.05

Fig. 32. Behavior detected by HomeCageScan

Fig. 33. Inducible expression of Bβ1 in PC12 cells by using a Complete control® Inducible Mammalian Expression System (Strategene)

(A) Ecdysone regulatory system overview. (B) pEGSH/Bβ1 contructs, the Bβ1 cDNA fragment (1.3kb) was subcloned into the multiple cloning site (MCS) of pEGSH vector. (C) Restriction map of pEGSH/Bβ1 contruct.

Lane 1, uncut; Lane 2, pEGSH/Bβ1 digested by XhoI/XbaI..

Gene of interest

Interested gene no expression

Interested gene expression Gene of interest

5’ 3’

XhoI XbaI

pERV3 receptor vector

B M 1 2

1.3kb C

Fig. 34. Cell morphology observation and MTT assay of cell viability.

(A) No difference was identified in cell morphology after PonA induction.

(B) No difference was identified in cell viability after PonA induction.

(scale bar: 30 µm) (mean±S.E.M.).*, p<0.05

Fig. 35. RNA expression of clonal cells after NGF treatment RT-PCR analysis of RNA expression level of clonal cells. The Bβ1

expression level was significantly higher than non-induction group. Actin expression is used as an internal control. Lane 1, 48 hr-PonA induction after NGF treatment for 48 hrs; Lane 2, No PonA induction after NGF treatment for 48 hrs; Lane 3, PonA induction 48 hrs; Lane 4,

non-induction of NGF and PonA. (B) quantitation of the results of RT-PCR analysis. (mean±S.E.M.).*, p<0.05 **, p<0.01

1 2 3 4 1 2 3 4 M 1 2 3 4 1 2 3 4 NGF 48hrs + EtOH 48hrs PonA 48hrs

Fig. 36. Western blot analysis for identification of the Bβ1 overexpression in the cells after PonA induction.

Flag antibody was used to detect overexpression of Bβ1 protein in clonal cells. (A) after PonA induction in low serum condition, and (B) Lane 1, 48 hr-PonA induction after NGF treatment for 48 hrs; Lane 2, No PonA induction after NGF treatment for 48 hrs; Lane 3, PonA induction 48 hrs;

Lane 4, non-induction of NGF and PonA. There was no leaky expression after PonA treatment. (C) is quantitation of A; (D) is quantitation of B (50 µg protein was loaded in each lane) (mean±S.E.M.).*, p<0.05 **, p<0.01

clone3 ^flag

Fig. 37. ICC analysis of clonal cells after 48 hr-PonA induction following NGF treatment for 48 hrs.

ICC analysis using flag antibody showed strong homogeneous cytoplasmic staining after NGF treatment 48 hrs followed PonA induction for 48 hrs. (scale bar: 30 µm)

DAPI Flag Merge PonA

+ Clone 3

Clone 8

Fig. 38. ICC analysis of clonal cells after 48 hr-PonA induction under serum starvation for 48 hrs.

ICC showed homogeneous cytoplasmic staining of Bβ1 in serum starvation condition followed PonA induction for 48 hrs. (scale bar: 30 µm)

DAPI Flag Merge PonA

+ Clone 3

Clone 8

Fig. 39. ICC analysis of clonal cells after 48-hr normal condition following 48 hr-PonA induction.

ICC showed homogeneous cytoplasmic staining of Bβ1 in normal condition followed PonA induction 48hrs. (scale bar: 30 µm)

DAPI Flag Merge PonA

+ Clone 3

Clone 8

Fig. 40. MTT assay results of cell lines with Bβ1 overexpressed or pEGSH vector only

(A) Cell growth was characterized by MTT assay after different period of ponA induction. (B) Cell growth characterization by MTT assay for different period after 48 hr-PonA induction following NGF treatment for 48 hrs. (mean±S.E.M.).*, p<0.05

OD570nm

seeding

NGF PonA/EtOH

24hrs 48hrs 24hrs 24hrs 24hrs Dendrite measurement

24hrs

Fig. 41. Observation of cell morphology after NGF treatment

(A) Schemetic diagram of time scale of the experiments. (B) Microscopic images of cells. (C) Measurement of dendrite length of cells in different time point after NGF treatment. (D) Outgrowth spine numbers of cells at different time point after PonA induction following NGF treatment. (scale bar: 30 µm) (mean±S.E.M.).*, p<0.05

Average of outgrowth spine number

seeding

NGF PonA & oxidative drugs 24hrs 48hrs 48hrs

MTT assay

Fig. 42. MTT assay results of cell lines with Bβ1 overexpressed or pEGSH vector only after oxidative chemical treatment.

(A) Schemetic diagram of time scale of the experiments. The cell

viability measured after treatment for 48 hrs with (B) H2O2, (C) TBH, and (D) STS oxidative chemicals. (mean±S.E.M.).*, p<0.05

H2O2

在文檔中建立和分析過量表現PPP2R2B 基因Bβ1之細胞與基因轉殖小鼠 (頁 62-105)