Objective To knock out p21 gene in human malignant rhab doid tumor(MRT)cell line G401 by using CRISPR/Cas9 genome engineering technology. Methods The expression of p21 was detected by reverse transcription quantitative PCR (RT-qPCR) and Western blot assay in several MRT cell lines. The guide RNA was designed by targeting the third exon of p21 gene,which encoded its home domains, and then subcloned into lentiCRISPR v2 vector and validated sequencing. The validated plasmids were further used to package and produce the lentivirus in 293T cells, and the G401 cells were infected, then puromycin was used to screen positive cells, and the clusters of G401 monoclonal cells, were obtained by selecting monoclonal cells and culturing under the microscope. The RNA and protein of new clonal cell line were extracted, and RT-qPCR and Western blot assay were applied to confirm whether p21 was successfully knocked out. Results The p21 was highly expressed in MRT tumor cells. The CRISPR/Cas9 lentivirus plasmids, targeted p21 gene were successfully constructed. Compared with negative control group,the expression of p21 was not detected in G401 monoclonal cells, which were successfully screened. Conclusion In view of the difficult transfection of cells such as G401, p21 knockout stable cell line has been successfully constructed by using CRISPR/Cas9 system, which lays the foundation for further study of the mechanism of p21 in MRT tumors .

Objective:To understand the current status of iodine deficiency disorders in children aged 8 - 10 years in Beihai, Guangxi, and to provide evidence for prevention and treatment of the disease.Methods:According to the "National Iodine Deficiency Disorders Surveillance Program" (2016 edition) and the "2016 - 2018 Beihai Iodine Deficiency Disorders Surveillance Program", Beihai was divided into administrative regions by stratified multi-stage sampling method in 2016 - 2018. Each district and county was divided into five parts according to directions: east, west, south, north and middle. One primary school was selected in each direction and 40 non-boarding students aged 8 - 10 years were selected from each primary school. Urine samples and edible salt samples were collected from children to detect urinary iodine and salt iodine contents. Thyroid volume of children was determined by B-ultrasound method, and the rate of goiter was calculated.Results:A total of 800 children aged 8 - 10 years were investigated, including 200 in 2016, 200 in 2017, and 400 in 2018. The ratio of male to female was 1.08:1.00 (415/385); the median salt iodine of children aged 8 - 10 years was 19.7 mg/kg; the edible rate of qualified iodized salt was 54.4% (435/800). The median urinary iodine of children aged 8 - 10 years in Beihai was 154.3 μg/L. The median urinary iodine of children aged 8, 9 and 10 years were 182.0, 141.9, 163.3 μg/L, respectively. The median urinary iodine of boys (164.3μg/L) was higher than that of girls (140.0 μg/L, Z = - 3.765, P < 0.05); there was a difference in the median urinary iodine between children aged 8 - 10 years in different regions ( H = 33.621, P < 0.05), and Tieshangang District had the lowest (117.6 μg/L); the median urinary iodine of 314 people was in the appropriate range (100 - < 200 μg/L), accounting for 39.3% (314/800). The total thyroid goiter rate was 1.1% (9/800) in children aged 8 - 10 years; the thyroid goiter rate was different in children from different regions ( P < 0.05), and the thyroid goiter rate (3.5%, 7/200) in children aged 8 - 10 years in Haicheng District was higher than that in other areas. Conclusions:The iodine nutrition status of children aged 8 - 10 years in Beihai is appropriate. Beihai should continue to adhere to salt iodization to prevent and treat iodine deficiency disorders, and take measures to adapt to local conditions and accurately supplement iodine for children aged 8 - 10 years in different districts and counties.