This document describes the development of several in vivo experimental models for anti-cancer research including: 1) heterotransplant models using human tumor xenografts in mice to preserve the tumor microenvironment; 2) transgenic mouse models that overexpress oncogenes or knockout tumor suppressor genes; and 3) conditional knockout mouse models where genes can be conditionally deleted in specific tissues. Details are provided on generating transgenic mouse lines that overexpress a deleted form of DNMT3B and generating a conditional HuR knockout mouse line using a targeting vector with loxP sites. The goals are to have repeatable, systematic preclinical models to better evaluate drug efficacy.

1. Develop in vivoExperimental Therapeutic Model for Anti-Cancer Research Mark (Zhi-Qing) Ma, Ph.D.

3. Transgenic mouse mode (Over-expression of oncogene)

5. Tumor Genome Analysis Pre- and Post Treatment intact tumor microenvironment GeneChip Human Exon 1.0 ST Array DNA Methylation Analysis other genetic and epigenetic assays Genome-Wide Human SNP Array 6.0

6. matched matched 50 mouse models Repeatable Systematic Preclinical Efficacy Evaluation of Drugs (SPEED) CTRL Arm : 50 Pts Tx Arm : 50 Pts Pathak et al., Am J Trans Res, 2009

7. Transgenic Mouse Lines Over-expressing DNMT3B SPC/Δ3B4/IGFP (Δ3B4G) DrdI DrdI NdeI NotI NotI IRES-GFP pA+ SPC promoter ΔDNMT3B4 Geno#1 Forward Geno#1 Reverse Geno#2 Forward Geno#2 Reverse SPC/Δ3B4 del/IGFP (Δ3B4DG) DrdI DrdI NotI NdeI NotI IRES-GFP pA+ SPC promoter ΔDNMT3B4 del Geno#1 Forward Geno#1 Reverse Geno#2 Forward Geno#2 Reverse

8. Purification of Transgene fragments for Microinjectin SPC/Δ3B4Del/IGFP (Δ3B4G) SPC/Δ3B4/IGFP (Δ3B4G) Transgene 10 kb 8 6 4 3 Vector

9. Genotyping Strategy of Transgenic Mice Tail DNA + Δ3B4DG Tail DNA + Δ3B4DG Tail DNA + Δ3B4G Tail DNA + Δ3B4G 100 bp Marker 1 kB Marker Tail DNA Tail DNA Geno#1 Geno#2

10. Generation of Conditional Mutant Cre floxed breeding ATG Promoter Cre Exon3 Exon2 Tissue Specific Conditional KO mice

11. Generation of FloxedHuR Mouse Line 5 ARM (5.1KB) 3 ARM (1.7 KB) 5 ARM probe 3 ARM probe E E A A A A A A E E E E2 E1 E3 Neo PGK-TK Targeting Vector LoxP LoxP LoxP WT Mutant 5ARM (Apl I) 10.0kb 11.3kb 3ARM (EcoRI) 5.5kb 7.5kb