Holly’s Work Progress in 08-177

Dec 2008 - July 2009

Outline

• ELISA assays of 6 inbred strains of mice• 08-177 B6C3H F2 work progress

ELISA assays of 6 inbred strains of mice

Strains Quantity RANKL ELISA PTH ELISA

A/J 19 V V

129/SvimJ 20 V V

C57BL/6J 20 V V

C3H/HeJ 20 V V

CAST/Ei 20 V

NOD/LtJ 20 V

~35 inbred strains in plan, 17% completion

Plasma sRANKL ELISA Assays of Inbred Strains of mice

• At baseline sRANKL level varies among different strains: C3H and B6 are much higher than NOD/Ltj, A/J, 129/SvimJ, and CAST/Ei is the lowest

• Following 3wks 100ppm F treatment, only A/J see significant dose response decrease by 64% (p<0.01)

Plasma Intact PTH ELISA assays of inbred strains of mice

• At baseline, A/J and 129/SvimJ PTH level is higher than that of C3H and B6

• Following 3wks 100ppm F treatment, A/J sees dose response decrease by 31% (p<0.1), but no significant changes in B6, C3H or 129/SvimJ.

Serum Intact PTH ELISA Assays

0

50

100

1500PPM50PPM

*

* P<0.1

Seru

m in

tact

PT

H (

pg

/ml+

/-S

D)

• Serum PTH of F treated (50ppm, 60days) A/J is higher than that of untreated

Old F2 Genotyping Samples Preparation

• 187 genomic DNA samples prepared from previous F2 mice livers/spleens

• 140 of 187 samples sent to Jackson Lab in Jun2009

08-177 B6C3HF2 work progressWork Number Completed

Breeding 309 (+ 2 pregnant F1 mice)

Genotyping samples preparation 212 (156 sent)

sRANKL ELISA assays 212

Intact PTH ELISA assays by Sep 09

B6C3H F2 Treatment PlanDOB1 DOW1 DOT1 DOD Total mice Male Cage#F-

maleF-

maleCage# C-male

CTRL-male

Female Cage# F-female

F-female Cage# C-female

CTRL-Fem.

Male cage

Female cage

Jan 10, 2009 Feb 2, 2009 Feb 23, 2009 Mar 16, 2009 8 2610047 2    6610048 3610049 31 2

Jan 10, 2009 Feb 2, 2009 Feb 23, 2009 Mar 16, 2009 7 1610047 1    6610055 3610056 31 2

Jan 10, 2009 Feb 2, 2009 Feb 23, 2009 Mar 16, 2009 6 2    610065 2 4   0610066 41 1

Jan 10, 2009 Feb 2, 2009 Feb 23, 2009 Mar 16, 2009 4 3610059 3    1   0610066 11 1

Jan 10, 2009 Feb 2, 2009 Feb 23, 2009 Mar 16, 2009 7 0        7610057 3610058 40 2

Jan 16, 2009 Feb 2, 2009 Feb 23, 2009 Mar 16, 2009 8 5    610043 5 3610044 3  01 1

Jan 10, 2009 Feb 2, 2009 Feb 23, 2009 Mar 16, 2009 8 5610063 5    3610064 3  01 1

Jan 10, 2009 Feb 2, 2009 Feb 23, 2009 Mar 16, 2009 8 3  0610045 3 5   0610046 51 1

Jan 10, 2009 Feb 2, 2009 Feb 23, 2009 Mar 16, 2009 6 4    610061 4 2610062 2   1 1

Jan 10, 2009 Feb 2, 2009 Feb 23, 2009 Mar 16, 2009 6 6610052 3610053 3 0        2 0

Jan 13, 2009 Feb 2, 2009 Feb 23, 2009 Mar 16, 2009 9 4610050 4    5610051 5  01 1

Feb 2, 2009 Feb 26, 2009 Mar 23, 2009 Apr 14, 2009 3 0        3     614378 3  1

Feb 2, 2009 Feb 26, 2009 Mar 23, 2009 Apr 14, 2009 9 5    614380 5 4     614379 41 1

Feb 4, 2009 Feb 26, 2009 Mar 23, 2009 Apr 14, 2009 6 6614376 3614377 3 0        2 0

Feb 5, 2009 Feb 26, 2009 Mar 23, 2009 Apr 14, 2009 8 3614382 3    5614381 5   1 1

4/16-18/2009 May 11, 2009 Jun 1, 2009 Jun 23, 2009 14 7629077 3629081 4

7629082 3

629076 42 2

4/16-18/2009 May 11, 2009 Jun 1, 2009 Jun 23, 2009 6 3629078 3   

3    629079 3 1 1

4/16-18/2009 May 11, 2009 Jun 1, 2009 Jun 23, 2009 4 0      

4   

629080 40 1

4/16-18/2009 May 11, 2009 Jun 1, 2009 Jun 24, 2009 5 2   629084 2

3629083 3    1 1

4/16-18/2009 May 11, 2009 Jun 1, 2009 Jun 24, 2009 11 7629087 3629085 4 4629086 4    2 1

4/16-18/2009 May 11, 2009 Jun 1, 2009 Jun 24, 2009 3 3    629088 3 0        1 0

4/23-27/2009 May 18, 2009 Jun 8, 2009 Jun 29, 2009 14 8629158 5629152 3 6629154 3629150 32 2

4/23-27/2009 May 18, 2009 Jun 8, 2009 Jun 29, 2009 12 4    629155 4 8629156 4629151 4 1 2

4/23-27/2009 May 18, 2009 Jun 8, 2009 Jun 29, 2009 7 4    629157 4 3     629153 31 1

4/29-30/2009 May 25, 2009 Jun 15, 2009 Jul 7, 2009 12 4630626 4   

8630627 3630625 51 2

4/29-30/2009 May 25, 2009 Jun 15, 2009 Jul 7, 2009 13 5630629 5    8630630 5630628 31 2

4/29-30/2009 May 25, 2009 Jun 15, 2009 Jul 7, 2009 7 3630632 3    4630631 4   1 1

Jul 5, 2009 Jul 27, 2009 Aug 17, 2009 Sep 7, 2009 16 62 33 3

101 56 52 2

Jul 5, 2009 Jul 27, 2009 Aug 17, 2009 Sep 7, 2009 12 85 57 3

4     4 42 1

Jul 5, 2009 Jul 27, 2009 Aug 17, 2009 Sep 7, 2009 3 3   8 3

0        1  

Jul 10, 2009 Jul 31, 2009 Aug 24, 2009 Sep 14, 2009 10 710 311 4

39 3   2 1

Jul 10, 2009 Jul 31, 2009 Aug 24, 2009 Sep 14, 2009 11 914 5 13 4

212 2    2 1

        0                                                              Total mice 263 132  66  66 131  66  65         Total cages   61   15  14    16  16 38 37% Female/total 50%  % F treatment 50%                       % F-female/ttl F 50% % F female/Total Female 50%        

% F male/ttl male 50%        

08-177 B6C3HF2 – Genotyping

• 212 genomic DNA samples prepared from 08-177 B6C3HF2 mice livers/Spleens

• 156 of 212 samples sent to HPCPG in July 09; the rest of 56 ready to be sent in Aug 09

08-177 B6C3HF2 – sRANKL ELISA Assays

See Hongyou’s presentation

Results & Discussion• At baseline, sRANKL level or intact PTH level varies among different

strains;

• Following 3 wks of 100ppm F treatment, different strains response differently. Among 6 inbred strains only A/J see significant dose response decrease of sRANKL by 64% (p<0.01) and decrease of intact PTH by 31% (p<0.1), which did not show the similar trend of previous results (Dong Y 2007) – different anesthesia? Serum vs Plasma? F in diet? different length of treatment?...

Future work

• Continue work on more inbred strains and B6C3HF2s• Mini study to check the impact of anaethesia

(Ketamine/Xylazine or Avertin) on serum or plasma PTH level using ALPCO PTH ELISA kit and verify the effect of fluoride treatment on PTH level.– 40 male C3H mice, 8 groups of 5.

Questions …?

Thanks!