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 andconfirmed by PCR and digesti on 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.

Fatores de risco para doença arterial coronariana em crianças e adolescentes filhos de coronariopatia jovens / Coronary risk factors in children of young coronary artery disease patients

Objetivo - verificar a prevalência dos fatores de risco (FR) em crianças e adolescentes filhos de coronariopatas. Métodos - em 280 filhos de coronariopatas jovens (< 55 anos), submetidos à cirurgia de revascularizaçäo miocárdica, foi determinada a frequência de desvios dos valores considerados ideiais para a faixa etária da colesterolemia total-CT, trigliceridemia-TG, HDL-C e LDL-C, glicemia, da pressäo arterial (PA), do peso corpóreo (através do índice de Newen-Goldstein-ING). Foram também verificas as frequências do hábito de fumar, e de alteraçöes eletrocardiográficas. Crianças de 2 a 12 anos formaram o grupo GA e adolescentes de 12 a 19 anos formaram o grupo GB. Eventuais associaçöes entre o FR e a influência dos fatores idade, sexo, peso corpóreo e tabagismo sobre as variáveis lipídicas também foram estudadas. Resultados - no conjunto estudado, 48,2 por cento e 44,6 por cento apresentaram respectivamente valores de CT e LDL-Cacima dos considerados ideais, sendo que 21,7 por cento e 26,1 por eto apresentavam valores indicativos de risco também para adultos. TG acima de 200 mg/dl ocorreur em 1,4 por cento da amostra e valores diminuídos de HDL-C em 16,8 por cento. O sobrepeso e obesidade estiveram presentes, respectivamente, em 13,1 por cento e 20,0 por cento (12,9 por cento e 31,4 por cento em GA e 13,2 por cento e 15,8 por cento em GB) e influenciaram os níveis de TG. Tabagismo ocorreu em 10,4 por cento; houve somente 3 casos de hipertensäo arterial. Näo foram encontradas anormalidades da liemia e nem alteraçöes eletrocardiográficas. Conclusäo - a investigaçäo reitera a necessidade de particular atençäo preventiva nos filhos de coronariopatas jovens, considerando a levada frequência de desvios do metabolismo lipídoco e de aumento do peso corpóreo (sobrepeso e obesidade)

Purpose - To determine whether children and adolescents, whose fathers have established coronary artery disease (CAD), have increased prevalence of coronary rishfactors (RF). Methods - The frequencies of abnormal values of lipid variables, glucose, blood pressure, obesity index (calculated through Newen-Goldstein index), smoking and electrocardiographic alterations (ECG), were assessed in 280 descendents of young revascularized patients (<55 years). The study population was divided in two groups according to age, respectivelly GA (2 to 12 years) and GB (12 to 19 years). Eventual influences ofage, gender, obesity and smoking on lipid variable were evaluated through variance analysis. Results - Of the study population, 48.2% and 44.6% had total cholesterol (TC) and LDL-C respectively above the desirable values; 21.7% and 26.1% had values similar to adults under increased risk. Triglyceridemia (TG) >200mg/dl was found in 1.4% of the sample and lower values of HDL-C in 16.8%. Overweight and obesity were observed in 13.1% and 20.0% of the patients and influenced TG levels in GB. Smoking occurred in 10.4%; hypertension in 3 cases and none had abnormal glucose levels or ECG. Conclusion - Healthy children of fathers with established CAD, exhibit a high freqüency of altered lipid profile and increased body weight. The results suggest the need for early identification of RF in offspring of young CAD patients, thus emphasizing changes in risk profile and improving lifestyle

Comparison of the effects of lab chow and casein diets based on body and brain development of rats

1. In order to study the effects of protein quantity and quality on development, Wistar rats were submitted to four different diets during lactation and post-lactation periods. Three isocaloric diets were utilized with 6 per cent (M), 16 per cent (W16) and 20 per cent (W20) of protein (casein), and the fourth diet (C) consisted of a commercial lab chow containing 22 per cent protein. 2. During the lactation and post-lactation periods the body weights of dams and pups were recorded weekly. On the 49th day of age (21 days of suckling and 28 days of ad libitum chow), all animals were sacrificed and the brains removed and weighed. 3. Dams from all groups increased food intake during the lactation period, but dams of the M group consumed a lower amount of diet as compared with other groups. Only the body weight of M dams was affected by diet during the lactation period, but the body weight of pups from the M and C groups was lower than in the other two groups. At 49 days of age C and M pups showed a significantly lower brain weight compared with W16 and W20 pups. 4. Thus, a commercial lab chow diet does not promote normal body and brain development as compared with balanced diets containing 16 or 20 per cent protein. These results emphasize the need for further studies in order to evaluate other biological and behavioral parameters that might be altered by a lab chow diet