Zhou Guo-ling, Song Wei, Fu Xiang-hui, Xin Li, Tang Xiao-bin, Feng Dong-xiao, Liu Guang, Liu De-pei
National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, CAMS and PUMC, Beijing 100005, China.
Zhongguo Yi Xue Ke Xue Yuan Xue Bao. 2007 Jun;29(3):293-301.
To establish chromosome conformation capture (3C) strategy and to use this method for exploring the effect of chromosome conformation on human alpha-globin gene expression in the human alpha-globin transgenic mouse.
Homozygous human alpha-globin transgenic male mouse was crossed with KM female mouse. The 14.5-day post-coitum (dpc) embryos were used for the isolation of fetal liver and fetal brain cells. Homogeneous single-cell suspension was treated with formaldehyde to crosslink the chromatin conformation in the nuclear. The cross-linked chromatin compound was digested with Nco I and then ligated with T4 DNA ligase. The ligated compound was reversely cross-linked and then the ligated genomic DNA was purified for PCR analysis. The primers were designed along the two sides of cut and ligated sites. Semi-quantitative PCR was used to analyze the chromosome conformation of the whole human alpha-globin gene locus in fetal liver and fetal brain cells.
When HS40 fragment was used as the fixed fragment, in fetal brain cells, the ligation frequencies of HS40 fragment with other fragments were decreased as the linear distances to HS40 fragment were increasing; while in fetal liver cells, two active genes (alpha1 and alpha2) fragments showed higher ligation frequencies with HS40 fragment than other fragments. However, the fragment containing an inactive gene (xi) displayed the comparable low ligation frequency as that in fetal brain. When alpha2 fragment was used as the fixed fragment, similarly, in fetal brain cells the ligation frequencies of alpha2 fragment with other ones were decreased as the linear distances increasing; when in fetal liver cells, it showed higher ligation frequencies with two upstream regulatory elements (HS 40 and 33). However, it showed a little bit lower ligation frequency with another two upstream regulatory elements (HS10 and 8) than those in fetal brain.
In fetal liver cells, the distant regulatory elements are in close proximity to the downstream of the expressed globin genes through looping out, the interval region; however, in fetal brain, they were not in vicinity to the expressed globin genes.
建立染色体构象捕获（3C）策略，并运用该方法探究染色体构象对人α-珠蛋白转基因小鼠中人α-珠蛋白基因表达的影响。
将纯合的人α-珠蛋白转基因雄性小鼠与KM雌性小鼠杂交。取妊娠14.5天（dpc）的胚胎用于分离胎儿肝脏和脑细胞。用甲醛处理均匀的单细胞悬液，使细胞核内的染色质构象交联。交联的染色质复合物用Nco I消化，然后用T4 DNA连接酶连接。连接的复合物进行反向交联，然后纯化连接的基因组DNA用于PCR分析。引物沿着切割和连接位点的两侧设计。采用半定量PCR分析胎儿肝脏和脑细胞中整个人α-珠蛋白基因座的染色体构象。
当HS40片段用作固定片段时，在胎儿脑细胞中，HS40片段与其他片段的连接频率随着与HS40片段线性距离的增加而降低；而在胎儿肝细胞中，两个活跃基因（α1和α2）的片段与HS40片段的连接频率高于其他片段。然而，含有一个不活跃基因（ξ）的片段显示出与胎儿脑细胞中相当的低连接频率。当α2片段用作固定片段时，同样地，在胎儿脑细胞中，α2片段与其他片段的连接频率随着线性距离的增加而降低；在胎儿肝细胞中，它与两个上游调控元件（HS40和33）显示出更高的连接频率。然而，它与另外两个上游调控元件（HS10和8）的连接频率比胎儿脑细胞中的略低。
在胎儿肝细胞中，远距离调控元件通过环出间隔区域与表达的珠蛋白基因下游紧密靠近；然而，在胎儿脑中，它们并不靠近表达的珠蛋白基因。
