Construction of engineered murine embryonic stem cells with conditional knockout of FGFR2 depending on Cre-loxP.

OBJECTIVE: To investigate the functions of Fibroblast Growth Factor Receptor-2 (FGFR2) at different stages of cell differentiation. The engineered murine embryonic stem (ES) cells with conditional knockout of FGFR2 were developed depending on Cre-loxP. METHODS: Cre-loxP system was used in a conditional targeting vector. The competent AM-1 bacteria, which expressed Cre-recombinase, was used to confirm the Cre-mediated deletion of the floxed exons 7 and 8 of FGFR2. The targeting vector was electroporated into the ES cells, and the transfected ES cells were screened with G418 and Ganciclovir. Finally, the ES clones with correct targeting events were identified by Southern Blot and PCR. RESULTS: The targeting vector with conditional knockout of murine FGFR2 was successfully constructed and confirmed by PCR and digestion analysis in bacteria. 86 ES clones were collected by selective culture with G418 and Ganciclovir. Four of the 86 ES clones were found containing the targeting gene sequence in genomic DNA proved by Southern Blot with a 5'-end flank probe. Two of the four ES clones had the correct targeting events that included the insertion of the targeting gene sequence in genomic DNA and were checked by Southern Blot with a 3'-end flanking probe. Finally, the insertion of loxP (loxP3) between exons 8 and 9 in genomic DNA was identified in one of the two ES clones by Southern Blot and PCR. CONCLUSION: FGFR2 conditional knockout depending on Cre-loxP can be successfully used in ES cells.

Extremity-preserving efficacy of vascular prosthesis grafting in repairing arterial injuries of extremities / 中国组织工程研究

BACKGROUND: Vascular injuries of the extremitiesy are frequently concomitant with vascular defects and are usually repaired by autologous vessel grafting. However, the source of autologous vessels is limited and the preparation of them is traumatic.OBJECTIVE: To retrospectively evaluate the extremity-preserving efficacy of vascular prosthesis grafting in repairing major vascular injuries of the extremities in 29 cases.DESIGN: A retrospective study.SETTING: Department of Orthopedics and Traumatic Surgery, Center for Battle Wound and Trauma of Chinese PLA, Research Institute of Surgery,Daping Hospital, Third Military Medical University of Chinese PLA.PARTICIPANTS: We selected 29 patients with major vascular injuries of the extremities repaired by vascular prosthesis grafting who received the treatment at the Department of Orthopedics and Traumatic Surgery of the Center for Battle Wound and Trauma of the Chinese PLA of the Research Institute of Surgery of, Daping Hospital of the Third Military Medical University of Chinese PLA between January 1989 and December 2000. There were 23 males and 6 females. Injury sites: 9 cases were at subclavian artery, 6 at axillary artery, 2 at brachial artery, 10 at femoral artery, 1 at femoral vein and 1 at popliteal artery. 11 of these 29 cases (37.9%) were complicated with shock, 8 with fractures and dislocations, 5 with peripheral nerve injuries and 3 with infections.METHODS: The vascular prosthesis was anastomosed end to end with the trimmed culprit vessels. As for the 3 cases with infective arterial injuries,the vessels were placed away from the infected region and were bridged together in a non-inf1ammatory region and then muscles or musculo-cutaneous flaps were used to cover the infected regions.The functions of the extremities of the patients were evaluated according to MAS at week 2 and 1 year after the operation.MAIN OUTCOME MEASURES: The vascular patency rate and the extremity-preserving outcomes..RESULTS: All 29 patients entered the stage of result analysis. Extremitypreserving efficacy: All the patients who underwent vascular prosthesis grafting had their extremities preserved. Only 1 patient had the late sequelae of ulcers in the foot sole due to extended extremity ischemia and nerve damage. The rate of 2-week patency rate of the grafted vessels was 100% and the 1-year rate of patency was 96.5%, as revealed by Doppler blood stream scanning. The excellent and good rate of the function of extremity was 89%.CONCLUSION: Vascular prosthesis grafting is the one of the methods for repairing the major vascular injuries so as to preserve the extremities and their functions.