Huo Zhong-jun, Wen Zong-hua, Lin Jing, Wang Kun, Huang Zhi-bin, Dai Zhao-xia, Ma Ning, Yan Guang, Chen Ying-hua, Chen Xiao-hui, Liu Wei, Ma Pin-yun, Luo Wei-hao, Zhao Ying, Fan Shu, Zhao Jia-jia, Huang Hong-hui, Wen Zi-long, Zhang Wen-qing
Department of Cell Biology, Southern Medical University, Guangzhou 510515, China.
Nan Fang Yi Ke Da Xue Xue Bao. 2010 May;30(5):931-5.
To screen and identify zebrafish mutants with erythropoiesis defects by N-ethyl-N-nitrosourea (ENU) mutagenesis and large-scale forward genetic screening using beta e 1 as the marker.
The chemical mutagen ENU was used to treat healthy wild-type male fish (AB strain, F0). The surviving ENU-treated fish were mated with wild-type female fish to generate F1, and further F2 family was generated by F1 family intercross. The adult F2 fish were intercrossed within each F2 family and the resulting F3 embryos from each crossing were subjected to whole mount in situ hybridization (WISH) with the beta e 1 probe. Mutagenesis was performed by treating the male zebrafish with ENU to induce mutations in pre-meiotic germ cells to generate the founders, which were outcrossed to obtained the F1 fish. The F1 fish from different founders were mated to generate the F2 families. F3 embryos from the sibling cross in the F2 family were examined by whole mount in situ hybridization using beta e 1-globin probe. The putative mutants were then characterized with different hematopoiesis markers.
We identified 4 beta e 1-deficient mutants with erythropoiesis defects, including two with specific erythiod lineage defects and two with concurrent lymphopoiesis defects.
通过N-乙基-N-亚硝基脲（ENU）诱变，并以βe1作为标记进行大规模正向遗传筛选，以筛选和鉴定具有红细胞生成缺陷的斑马鱼突变体。
使用化学诱变剂ENU处理健康的野生型雄鱼（AB品系，F0）。将存活的经ENU处理的鱼与野生型雌鱼交配产生F1，然后通过F1家系杂交产生F2家系。对成年F2鱼在每个F2家系内进行杂交，并对每个杂交产生的F3胚胎用βe1探针进行全胚原位杂交（WISH）。通过用ENU处理雄性斑马鱼，在减数分裂前的生殖细胞中诱导突变以产生奠基者，将其与野生型杂交获得F1鱼。来自不同奠基者的F1鱼相互交配产生F2家系。使用βe1-珠蛋白探针通过全胚原位杂交检查F2家系中同胞杂交产生的F3胚胎。然后用不同的造血标记对推定的突变体进行表征。
我们鉴定出4个具有红细胞生成缺陷的βe1缺陷突变体，其中2个具有特定的红系谱系缺陷，2个同时具有淋巴细胞生成缺陷。
