Steroid sulfatase deficiency is a common genetic disorder, with a prevalence of approximately one in every 3500 males world wide.About 90% of these patients have complete gene deletions, which appear to result from re...Steroid sulfatase deficiency is a common genetic disorder, with a prevalence of approximately one in every 3500 males world wide.About 90% of these patients have complete gene deletions, which appear to result from recombination between members of a low-copy repeat family (CR1-232 is the prototype) that flank the gene. RUI and RU2 are two VNTR elements found within each of these family members.The RU1 consists of 30bp repeating units and its length shows minimal variation among individuals. The RU2 element consists of repeating sequences which are highly asymmetric, with about 90% purines and no C's on one strand, and range from 0. 6kb to over 23kb among different individuals. We conducted a study to determine if the RU1 and RU2 elements can promote recombination in an in vivo test system.We inserted these elements adjacent to the neo gene in each of two pSV2neo derivatives. One of which has a deletion in the 5' portion of neo gene and the other having a deletion in the 3'portion. These plasmids were combined and used to transfect EJ human bladder tumor cells. Survival of cells in G418 indicates restoration of a functional neo gene by recombination between two deletion constructs. Thus counting G418 resistant colonies gives a quantitative measure of the enhancement of recombination by the inserted VNTR elements.The results showed no effect on recombination by the inserted RU1 element(compared to the insertion of a nonspecific sequence), while the RU2 element stimulated.recombination by 3. 5-fold (P< 0.01). A separate set of constructs placed RU1 or RU2 within the nitron of an exon trapping vector. Following transfection of cells, recombination events were monitored by a quantitative PCR assay that detected the approximation of primer banding sites (as a result of recombination).These studies showed that, as in the first set of experiments, the highly variable RU2 element is capable of stimulating somatic recombination in tnammalian cells.展开更多
常规经基因修饰腺病毒转染细胞后,即进行细胞的移植,并不对病毒转染效果进行检验。本研究采用HIF-1α与EGFP双基因重组腺病毒转染大鼠心肌干细胞,察看基因重组腺病毒是否能够转染心肌干细胞内;研究经HIF-1α与EGFP(enhanced green fluor...常规经基因修饰腺病毒转染细胞后,即进行细胞的移植,并不对病毒转染效果进行检验。本研究采用HIF-1α与EGFP双基因重组腺病毒转染大鼠心肌干细胞,察看基因重组腺病毒是否能够转染心肌干细胞内;研究经HIF-1α与EGFP(enhanced green fluorescence protein,EGFP)双基因重组腺病毒转染心肌干细胞后HIF-1α基因的表达情况。在荧光显微镜下,去察看病毒转染细胞的情况,确定病毒最适MOI值;采用实时定量PCR(real-time quantitative PCR,RT-qPCR)评价HIF-1α基因表达变化;应用Western blotting方法检测HIF-1α蛋白表达变化,进而获得良好的实验结果。展开更多
文摘Steroid sulfatase deficiency is a common genetic disorder, with a prevalence of approximately one in every 3500 males world wide.About 90% of these patients have complete gene deletions, which appear to result from recombination between members of a low-copy repeat family (CR1-232 is the prototype) that flank the gene. RUI and RU2 are two VNTR elements found within each of these family members.The RU1 consists of 30bp repeating units and its length shows minimal variation among individuals. The RU2 element consists of repeating sequences which are highly asymmetric, with about 90% purines and no C's on one strand, and range from 0. 6kb to over 23kb among different individuals. We conducted a study to determine if the RU1 and RU2 elements can promote recombination in an in vivo test system.We inserted these elements adjacent to the neo gene in each of two pSV2neo derivatives. One of which has a deletion in the 5' portion of neo gene and the other having a deletion in the 3'portion. These plasmids were combined and used to transfect EJ human bladder tumor cells. Survival of cells in G418 indicates restoration of a functional neo gene by recombination between two deletion constructs. Thus counting G418 resistant colonies gives a quantitative measure of the enhancement of recombination by the inserted VNTR elements.The results showed no effect on recombination by the inserted RU1 element(compared to the insertion of a nonspecific sequence), while the RU2 element stimulated.recombination by 3. 5-fold (P< 0.01). A separate set of constructs placed RU1 or RU2 within the nitron of an exon trapping vector. Following transfection of cells, recombination events were monitored by a quantitative PCR assay that detected the approximation of primer banding sites (as a result of recombination).These studies showed that, as in the first set of experiments, the highly variable RU2 element is capable of stimulating somatic recombination in tnammalian cells.
