PDF《Research Communication》

s Republic of China. Tel: 186-431- 85099170;

Fax: 186-431-85099285. E-mail: [email protected] Received

7 August 2010;

accepted

5 October

2010 ISSN 1521-6543 print/ISSN 1521-6551 online DOI: 10.1002/iub.389 IUBMB Life, 62(11): 819C824, November

2010 targeted ES clones. Chimeras were generated by microinjection of targeted ES cells into C57/BL6 blastocysts and blastocysts were carried to term by pseudopregnant females (17). Beta- Actin-tetR-Krab transgenic construct was built by ligation of Chicken Beta-Actin promoter to tetR-Krab cDNA (18, 19). Beta-Actin-tetR-Krab mouse (bAtKtg ) was generated by pronu- clei injection using Narishiga micromanipulator and Olympus inverted microscope. Founders were crossed with VEGFtetO mice on doxycycline food (doxy chow) and selected for germline transmission. Further crosses between double transgenic off- spring (VEGFtetO/1 /bAtKtg/1 ) generated mice homozygous for VEGFtetO alleles and bAtKtg alleles (VEGFtetO/tetO /bAtKtg/1 ). For experiment, VEGFtetO/tetO /bAtKtg/1 mice were removed of doxy chow at weaning age and switched to regular chow for at least

6 weeks before analysis (Marked by 2Dox). Control group was kept on doxy chow to the time of assay (Marked by 1Dox). All the mice were mixed littermates and assayed at the age of 8C10 weeks. Real-Time PCR VEGF expression was measured by real-time PCR after 6C8 weeks of doxycycline removal. Total RNA was extracted using RNAisoTM Plus (TAKARA, D9108A, Japan). One microgram of total RNA was reverse transcribed using the Reverse Transcription Systems from TAKARA. Real-time PCR was performed using SYBR Green (TOYOBO, QPK-201, Japan) and ROCHE Light Cycler 480. VEGF forward primer

50 -TGTACCTCCACCATGC CAAGT and reverse primer

50 -TGGTAGAC-GTCCAT GAACTTG were used. For template loading control, 18S forward primer

50 -CGCCGC-TAGAGGTGAAATTC and reverse primer

50 -CGAACCTCCGACTTTCGTTCT were used. Bleeding Time and Clotting Time Bleeding time was measured by tail-bleeding assay. Mice were anesthetized and the distal portion of the tails (3 mm from tip of the tails) were cut with razor blade and tails immersed in Figure 1. Repression of VEGF expression at mRNA level was........