4T1 BALB /c Western GFP. Establishment of a mouse mammary cancer model stably expressing human HER2 / neu gene and luciferase gene

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1 Mil Med Sci Vol 36 No 2 Feb neu neu neu 4T1 G418 Western neu 4T1 BALB /c G418 4T1 Western neu GFP 4T1 neu neu R A Establishment of a mouse mammary cancer model stably expressing human neu gene and luciferase gene WU Ting 1 TAN Ying-xia 2 WANG Ying-li 2 LI Su-bo 2 BAO Guo-qiang 2 JI Shou-ping 2* 1. Guangxi Medical University Nanning China 2. Institute of Transfusion Medicine Academy of Military Medical Sciences Beijing China * Corresponding author Tel jishouping@ yahoo. com Abstract Objective To establish a mouse breast cancer model stably expressing human neu and firefly luciferase gene. Methods 4T1 cells were successively transfected with human neu and firefly luciferase gene and selected with G418 and hygromycin respectively. The neu and luciferase protein in selected clones were detected by Western-blot and bioluminescence imaging technology. Finally the T1 cells stably expressing neu and luciferase gene were transplanted in the mammary fatpad of BALB / c mice. The proliferation and metastasis of the tumor graft were observed and determined with bioluminescence imaging technology. Results and Conclusion After transfection and selection G418 and hygromycin resistant 4T1 clones were obtained. Western blot analysis showed the transfected 4T1 cells expressed a high level of neu protein. FACS results showed that almost all the cells from the selected clone expressed GFP protein. Tumor graft experiments showed that the transfected cells had similar in vivo tumorigenesis and metastasis to wild-type 4T1 cells. The results suggest that an neu- and luciferase-expressing mouse breast cancer model is successfully established. Key words breast neoplasms transfection neu luciferases firefly fluorescein 7% ~ 10% 40% neu 1 2 neu neu neu 4 ~ 6 4T1 BALB /c Tel jishouping@ yahoo. com neu luciferase 4T1 neu neu

2 Mil Med Sci Vol 36 No 2 Feb ~ 20 d ~ 8 PBS 18 ~ 20 g BALB /c SPF 4T1 4T1-Luc SKBR neu pcdna3. 1-Luciferase 4T1-Luc pcdna3. 1-HYGRO-EGFP Lipofectamine TM RPMI 1640 pcdna3. 1-HYGRO-EGFP-neu 4T1- Gibco G418 HYGRO Amresco TRIzol Lipofectamine 2000 Invitrogen PEI Polysciences DNA NEB HindⅢ NheⅠ T4 DNA r /min 3 min TaKaRa Western neu neu GFP Promega -neu CST DNA 4T1-Luc-Her2 / neu DNA DL2000 Plus 4T1-Luc-Her2 /neu Nest T1 5 ~ 6 d SKBR-3 10% RPMI 2 cm μl % CO neu TRIzol SKBR-3 RNA 2 RNA Promega 25 5 min min neu min PCR HER2 /neu cdna 5'-GCGCTAGCATGGAGCTG- GCGGCCTT-3' 5'-CGAAGCTTCACTG- GCACGTCCAGACC-3' 98 1 min 98 G /ml μl μl Luc 400 μg /ml 15 ~ 20 d 100 μl Western neu GFP Vigorous DNA Omega neu PBS /ml BALB /c 150 μl /9 min SKBR-3 RNA 3 RNA 1 5 s s s min PCR neu cdna pcdna3. 1-HYGRO-EGFP HindⅢ NheⅠ PCR 7 1 SKBR-3 RNA T1 1. DNA DL2000plus 2. RNA 4T PEI RNA cdna pcdna3. 1-Luciferase 8 PCR PCR 2 μl 4T1 G μg /ml 3. 8 kb

3 Mil Med Sci Vol 36 No 2 Feb Her2 /neu cdna Her2 /neu cdna 2 4 4T1 2 neu PCR 1. DNA DL2000plus 2. PCR neu 2 HER2neu 5 PCR pcdna3. 1-HYGRO- EGFP DH5α DNA 4 3 neu Western M. SKBR-3. SKBR-3 4T1. 4T T1-Lu neu 4T1-Luc-neu 2 neu Western neu pcdna3. 1-HYGRO-EGFP- neu 4T1-Luc GFP GFP 100% 6 HER2 /neu-gfp 4T1- Luc- neu 3 neu pcdna3. 1-HYGRO-EGFP M. DNA DL2000plus 1 ~ T1 4T GFP A. 4T1-Luc B. 4T1-Luc-neu / 2. 4 neu D1 4T1-Luc-neu BALB /c a b 4T1-Luc V V = 1 /2 a b neu 16 d pcdna3. 1-HYGRO-EGFP- 2 cm 3 4T1 7 neu 4T1-Luc 2 cm 3 neu 200 μl 1 min SKBR-3 4T1 Western neu

4 Mil Med Sci Vol 36 No 2 Feb T1 8 3 BALB /c neu neu 90% 14 Taylor 15 neu HER2 /neu Neu neu B104 Neu neu neu neu B104 4T1 neu neu neu 4T1 4T1 neu neu cdna 4T1 neu neu neu cdna cdna 40% 4T1 Western 4T1- neu Luc-neu neu 1 neu 2 SKBR-3 4T1 neu -neu neu neu 3 4T1 neu HER2 /neu pl neu 4T1 MAPK 4T1-Luc-neu STAT PLCγ PI3K /AKT GFP 4 ~ FDA neu trastuzumab 4T1 Herceptin neu 9 neu DNA neu 10 ~ neu 13 neu 140

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