Questions to Think About When Completing Animal Costings

QUESTIONS TO THINK ABOUT WHEN COMPLETING BRR ANIMAL COSTINGS:

(See example answers at the end of this document to help with completion)

1. Which funder/s is this grant being submitted too?

Arthritis Research UK

2. When do you expect the grant to start?

1st July 2015

3. Number of months of the grant?

36 months

4. Ball-park figure (£) for all animal work you would like to request in relation to the total grant request – animal work can be as much as 45% of the total request for an in vivo dominant application.?

Would like £2,000 per month to cover bills; maximum £80,000.

BREEDING

5. How many different strains are to be used in the application, please give the strain short name and details of how you would like them each to be bred e.g. Hom cross, Het cross to WT etc… ?

1. Col2a1-cre mouse strain: B6;SJL-Tg(Col2a1-cre)1Bhr/J (Jackson Laboratory) 2. Floxed CB2 mouse strain (generated at the IGMM using CRISPR/Cas9 system) 3. Conditional CB2 KO mouse strain in chondrocytes and their controls (flox CB2 mouse strain) both from crossing strain1. with strain 2.

6. For each strain give the number of breeding cages you would like to hold each week and the length of time, in weeks you would like the breeding to continue for, e.g. 15 cages for 52 weeks per year over two years = 15x52x2 = 1560 cage weeks

For strain 1. = 8 cages for 52 weeks over 1 year = 416 cage weeks For strain 2. = 8 cages for 52 weeks over 1 year = 416 cage weeks For strain 3. = 8 cages for 52 weeks over 3 years = 1248 cage weeks

PURCHASE

7. If a strain is to be bred against a WT line or a reporter line, please give the background of each line and the number of WT animals that you would like to purchase each year for breeding

1. We will backcross the col2a1-Cre line (strain 1.) eight (8) times on C57BL/6 background (use up to 8 C57BL/6 female mice).

09a3baedd186050208e53b1933a78fec.doc 2. We will use a ROSA26 Cre reporter mouse line to cross them with strain 1. to validate the expression of col2a1 (purchase a pair of B6.129S4-Gt(ROSA)26Sortm1Sor/J strain from The Jackson Laboratory).

8. Please identify if any of the strains are to be NEW imports.

Strain 1. is new import.

EXPERIMENTAL

9. Give the number, age, weight and sex, of experimental animals needed each year from each strain. Give consideration to the following:-

a. Provide a short description and # for each expt. Number and type of each strain of animal, are they all the same strain, in-bred, out-bred, transgenic e.g. Balb/c from a supplier or are they from an in-house colony of transgenics already at UoE b. Age and / or weight at start of expt? c. Duration of the expt. In weeks, including lead-in time to get animals to correct start age / weight? d. Is a defined Sex required for the experimental cohorts? e. Will specialised caging be required e.g. IVC, Isolators, Metabolic caging.

Year 1

Exp1. Generate the floxed CB2 mouse strain using CRISPR/Cas9 system in C57BL/6 mice. Final background C57BL/6.

Exp2. Cross col2a1-cre mouse strain with the ROSA26 Cre reporter mouse line to validate the expression of col2a1.

Exp3. Backcross col2a1-Cre mouse in C57BL/6 at least 8 times. Final background C57BL/6.

Exp4. Adult male wild type, col2a1-cre mice and floxed CB2 mouse (all on a C57BL/6 background) at 8 weeks of age will be subjected to destabilisation of medial meniscus for 8 weeks. They will be sacrificed when they are 16 weeks of age and knee joints will be used. 10 mice per group, total N=30.

Year 2

Exp5. Cross col2a1-cre mouse strain with the floxed CB2 mouse strain to generate conditional CB2 KO mouse strain in chondrocytes (strain 3.). N=10 from each of strain (total N=20 both in the same C57BL/6 background).

Exp6. Adult male conditional CB2 KO mice in chondrocytes and control (floxed CB2 mouse strain) mice at 8 weeks of age will be subjected to destabilisation of medial meniscus with or without CB2 agonist treatment for 8 weeks. They will be sacrificed when they are 16 weeks of age and knee joints will be used (13 mice per group for the experiment without treatment [total 26], 10 mice per group for the experiment with vehicle treatment or CB2 agonist treatment [10x4 = 40]). Together total N=66

09a3baedd186050208e53b1933a78fec.doc Year 3

Exp7. Adult male conditional CB2 KO mice in chondrocytes and control (floxed CB2 mouse strain) mice at 9 and 12 weeks will be sacrificed and femoral heads for in vitro experiments. 10 mice from each strain for each time point. N=40.

10.What procedures do you wish to be preformed, please indicate those carried by you and those you wish carried out by BRR staff e.g. schedule 1 which includes carcass waste disposal, ear notching for identification / genotyping. Recovery or non-recovery surgery, injections, gavage, weight recording, behavioural studies, MRI, CT, PET, ultrasound etc...?

All procedures mentioned above will be carried out by me apart from some Schedule 1 procedures such as carcass waste disposal and ear notching.

11.It is strongly recommended that all unique strains used in a project which spans more than two years be cryopreserved via embryo or sperm freezing, the total application cost should reflect this.

Please include in cost sperm freezing for two lines with IVF quality control.

09a3baedd186050208e53b1933a78fec.doc Example Answers

QUESTIONS TO THINK ABOUT WHEN COMPLETING ANIMAL COSTINGS:

1. Which funder/s is this grant being submitted too? MRC

2. When do you expect the grant to start? NOV 2011

3. Number of months of the grant? 36

4. Ball-park figure (£) for all animal work you would like to request in relation to the total grant request – animal work can be as much as 45% of the total request for an in vivo dominant application.? Would like c.£2k per month to cover bills max c.£80K

BREEDING 5. How many different strains are to be used in the application, please give the strain short name and details of how you would like them each to be bred e.g. Hom cross, Het cross to WT etc… ? i. Jackson Laboratories STOCK Cftrtm1UncTg(FABPCFTR)1Jaw/J ii. HGU CF mouse, eggs from Julia Dorin, Edinburgh iii. G480c CF mouse, eggs from Julia Dorin, Edinburgh iv. S100 A9 KO mouse (B6 background), from Johannes Roth, University of Munster v. Backcross of Cftrtm1Unc with S100 A9 KO mouse vi. WT b6 mice

6. For each strain give the number of breeding cages you would like to hold each week and the length of time, in weeks you would like the breeding to continue for, e.g. 15 cages for 52 weeks per year over two years = 15x52x2 = 1560 cage weeks

For strains I, ii and iii = 2 cages for 52 weeks over 1st year = 104 cage weeks.

For all other strains = 5 cages for 52 weeks over all 3 years. = 780 cage weeks. PURCHASE 7. If a strain is to be bred against a WT line or a reporter line, please give the background of each line and the number of WT animals that you would like to purchase each year for breeding

8. Please identify if any of the strains are to be NEW imports.

i and iv are new imports

EXPERIMENTAL 9. Give the number, age, weight and sex, of experimental animals needed each year from each strain. Give consideration to the following:-

a. Provide a short description and # for each expt. Number and type of each strain of animal, are they all the same strain, in-bred, out-bred, transgenic e.g. Balb/c from a supplier or are they from an in-house colony of transgenics already at UoE b. Age and / or weight at start of expt? c. Duration of the expt. In weeks, including lead-in time to get animals to correct start age / weight? d. Is a defined Sex required for the experimental cohorts? e. Will specialised caging be required e.g. IVC, Isolators, Metabolic caging. 09a3baedd186050208e53b1933a78fec.doc Year 1

Exp 1. WT B6 adult males at 8 weeks old will be sacrificed and bones used, n=18.

Exp2. WT B6 adult females at 6 weeks old will be infected by intranasal instillation with bacterial suspension. Sacrifice at 24, 48 and 72 hrs. 3 different bacteria 6 mice in each group plus 6 controls for each time point n=68

Year 2

Exp3. S100 A9 KO (from Thomas Vogl, Munster, will supply 2 breeding pairs) please establish and expand the colony. As per Year 2, Mice infected by intranasal instillation with bugs. Sacrificed at 24, 48 and 72 hrs. 2 different bacteria 6 mice in each group plus PBS control. 6 controls for each time point and 2 different bacteria. N =54 KO, 54 WT controls (b6). N=108 animals in total.

Exp 4. ACUTE: Cftrtm1UncTg(FABPCFTR)1Jaw/J (n=6), HGU CF (n=6), G480c CF (n=6) infected by intranasal instillation and sacrificed at 24, 48 and 72 hrs. 6 controls at 3 time points. n=54 UNC, HGU, G480c (162). n=54 WT controls (b6) n=216 animals in total.

Year 3

Exp 5. CHRONIC: Cftrtm1UncTg(FABPCFTR)1Jaw/J (n=12), HGU CF (n=12), G480c CF (n=12), controls n=12. Treated daily with neb bugs (2 bugs plus PBS con) for 30 days then sacrificed. Therefore n= 36 UNC,HGU, G480c (108). N=36 WT controls (b6). N = 144 animals in total.

Exp 6. Backcross of Cftrtm1UncTg(FABPCFTR)1Jaw/J and S100 A9 KO. 6 generations then genotype.

Acute and chronic protocols for infection Double KO vs. UNC KO, S100 A9 KO and WT controls. Acute protocol as in exp 4. N=54 in each group. N = 216 animals in total. And a Chronic protocol n= 36 in each group. N=144 animals in total.

10.What procedures do you wish to be preformed, please indicate those carried by you and those you wish carried out by BRR staff e.g. schedule 1 which includes carcass waste disposal, ear notching for identification / genotyping. Recovery or non-recovery surgery, injections, gavage, weight recording, behavioural studies, MRI, CT, PET, ultrasound etc...? See above

11.It is strongly recommended that all unique strains used in a project which spans more than two years be cryopreserved via embryo or sperm freezing, the total application cost should reflect this.

Yes include in cost as sperm freezing for two lines with IVF quality control.

09a3baedd186050208e53b1933a78fec.doc