Objective To investigate the effect of the glutathione peroxidase 4(GPx4)on mouse so-matic cell reprogramming.Methods To compare the expressions of GPx4 in OG2 mouse embryonic fibroblast(OG2-MEF)cells(MEFs group)and mouse embryonic stem cells(mESC,mESCs group),the expression lev-el of intracellular GPx4 was determined by transcriptome sequencing technique and Western blot.To verify the effect of GPx4 on the efficiency of the somatic cells reprogramming,the complete open reading frame se-quence of GPx4 gene and its selenocysteine insertion sequence(SECIS)were connected to the retroviral vector pMXs for constructing the overexpressed plasmid pMXs-GPx4.Gpx4-targeting short hairpin RNA(shRNA)was synthesized and connected to pSUPER vector,GPx4 shRNA1 and GPx4 shRNA2 were constructed to knockdown GPx4 expression.The above plasmids were co-transfected with pMXs-Sox2,pMXs-Klf4 and pMXs-Oct4 into MEF cells for reprogramming induction to obtain the pMXs no-load control group(pMXs NC),pMXs GPx4 group,pSUPER no-load control group(pSUPER NC),GPx4 shRNA1 group and GPx4 shRNA2 group.The expressions of GPx4 gene and multifunctional marker genes Rex1,Sox2,Dappa3,Sall4,Oct4 and Nanog were detected by real-time fluorescence quantitative PCR.The induced pluripotent stem cells(iPSC)were detected by immunofluorescence staining;the number of iPSC clones generation was detected by alkaline phosphatase staining of pluripotent stem cells;the GPx4 protein expression was detected by Western blot.Results The mRNA and protein expression of GPx4 in the mESCs group was higher than that in the MEFs group;compared with the pMXs NC group,the expression level of GPx4 mRNA in the pMXs GPx4 group was significantly increased;compared with the pSUPER NC group,the GPx4 mRNA and protein levels in the GPx4 shRNA1 group and GPx4 shRNA2 group were decreased(P＜0.05);the iPSC clone number in the pMXs GPx4 group was higher than that in the pMXs NC group,but the difference was not statistically significant(P＞0.05).The number of iPSC clones in the GPx4 shRNA1 group and GPx4 shRNA2 group was significantly lower than that in the pSUPER NC group,and the difference was statistically significant(P＜0.05).After completing the reprogramming,compared with the original MEF cells,the expression levels of various pluripotent marker genes Rex1,Sox2,Dappa3,Sall4,Oct4 and Nanog in the generated iPSC of each group were increased.Conclusion GPx4 knockdown could inhibit the efficiency of somatic cell reprogram-ming,its generated induced pluripotent stem cells have the normal pluripotent gene expression ability.

Objective:To explore the diagnostic value of chromosome karyotype analysis, chromosomal microarray analysis (CMA) and whole exome sequencing (WES) in microcephaly.Methods:A total of 9 cases of microcephaly fetuses diagnosed by prenatal ultrasound or children with microcephaly diagnosed after birth were selected from the Sixth Affiliated Hospital of Guangzhou Medical University from January 2014 to August 2022.Karyotype analysis and/or CMA were used to detect. The cases with negative karyotype analysis and CMA results were further sequenced by trio-based WES (Trio-WES). Then the coding genes contained in the pathogenic copy number variation (CNV) fragments were analyzed by gene ontology (GO) enrichment. The genes related to the development of the central nervous system contained in the pathogenic CNV and the pathogenic genes found by Trio-WES were combined for gene interaction network analysis.Results:In this study, 9 cases of microcephaly were recruited, with the time of diagnosis ranged from 23 weeks of gestation to 7 years after birth, and the head circumference of fetus or children ranged from 18.3 to 42.5 cm (-7SD to -2SD). Karyotype analysis was detected in all 9 cases and no abnormality result was found. Eight cases were detected by CMA, and one abnormal was found. Five cases were detected by Trio-WES, and two cases were detected with likely pathogenic genes. The GO enrichment analysis of the coding gene in the 4p16.3 microdeletion (pathogenic CNV) region showed that: in biological process, it was mainly concentrated in phototransduction, visible light; in terms of molecular function, it was mainly concentrated in fibroblast growth factor binding; in terms of cell components, it was mainly concentrated in rough endoplasmic reticulum. Gene interaction network analysis suggested that CDC42 gene could interact with CTBP1, HTT and ASPM gene.Conclusions:CMA could be used as a first-line detection technique for microcephaly. When the results of chromosome karyotype analysis and/or CMA are negative, Trio-WES could improve the detection rate of pathogenicity of microcephaly.