Culture parameters for stable expansion, genetic modification and germline transmission of rat pluripotent stem cells.
Culture parameters for stable expansion, genetic modification and germline transmission of rat pluripotent stem cells.
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DOI：
10.1242/bio.2011029
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发表时间：
2012-01-15
期刊：
影响因子：
2.4
通讯作者：
Smith AG
中科院分区：
文献类型：
--
作者：
Blair K;Leitch HG;Mansfield W;Dumeau CÉ;Humphreys P;Smith AG
培养的多能性细胞对嵌合动物生殖系的贡献是其用于基因操作的必要条件。自大鼠胚胎干细胞首次被报道以来的三年里，从这种新资源中预期的转基因大鼠模型的增殖尚未实现。培养不稳定、核型异常和菌株变异被认为是造成种系定植能力差的原因。这些问题的解决对于将基于多能细胞的基因操作技术在大鼠中达到在小鼠中达到的效率水平至关重要。最近的报道描述了维持大鼠胚胎干细胞的各种替代方法，包括提供额外的小分子和选择性传代方法。相比之下，我们报道了整倍体，种系能力大鼠ES和胚胎生殖（EG）细胞系可以通过简单的贴壁培养方法在添加有丝分裂原激活蛋白激酶（ERK1/2）级联和糖原合成酶激酶3的原始两种抑制剂（2i）和细胞因子白血病抑制因子（LIF）的确定培养基中维持。我们展示了大鼠ES和EG细胞系的基因修饰、克隆扩增和通过种系的传播。我们还描述了当SD菌株囊胚用作DA或SD多能干细胞的受体时，对足月嵌合体的贡献有明显的偏好。
The ability of cultured pluripotent cells to contribute to the germline of chimaeric animals is essential to their utility for genetic manipulation. In the three years since rat embryonic stem (ES) cells were first reported the anticipated proliferation of genetically modified rat models from this new resource has not been realised. Culture instability, karyotypic anomalies, and strain variation are postulated to contribute to poor germline colonisation capacity. The resolution of these issues is essential to bring pluripotent cell-based genetic manipulation technology in the rat to the level of efficiency achieved in the mouse. Recent reports have described various alternative methods to maintain rat ES cells that include provision of additional small molecules and selective passaging methods. In contrast, we report that euploid, germline competent rat ES and embryonic germ (EG) cell lines can be maintained by simple adherent culture methods in defined medium supplemented with the original two inhibitors (2i) of the mitogen-activated protein kinase (ERK1/2) cascade and of glycogen synthase kinase 3, in combination with the cytokine leukaemia inhibitory factor (LIF). We demonstrate genetic modification, clonal expansion and transmission through the germline of rat ES and EG cell lines. We also describe a marked preference for full-term chimaera contribution when SD strain blastocysts are used as recipients for either DA or SD pluripotent stem cells.
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影响因子：
64.8
作者：
Skarnes WC;Rosen B;West AP;Koutsourakis M;Bushell W;Iyer V;Mujica AO;Thomas M;Harrow J;Cox T;Jackson D;Severin J;Biggs P;Fu J;Nefedov M;de Jong PJ;Stewart AF;Bradley A
通讯作者：
Bradley A
影响因子：
2.5
作者：
OUHIBI, N;SULLIVAN, NF;CLARKE, NJ
通讯作者：
CLARKE, NJ
影响因子：
3.7
作者：
Vassilieva, S;Guan, KM;Wobus, AM
通讯作者：
Wobus, AM
影响因子：
3.7
作者：
Hamanaka S;Yamaguchi T;Kobayashi T;Kato-Itoh M;Yamazaki S;Sato H;Umino A;Wakiyama Y;Arai M;Sanbo M;Hirabayashi M;Nakauchi H
通讯作者：
Nakauchi H
影响因子：
64.5
作者：
Ding, S;Wu, XH;Xu, T
通讯作者：
Xu, T