A recessive genetic screen for host factors required for retroviral infection in a library of insertionally mutated Blm-deficient embryonic stem cells.
A recessive genetic screen for host factors required for retroviral infection in a library of insertionally mutated Blm-deficient embryonic stem cells.
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在插入插入的BLM缺陷型胚胎干细胞库中逆转录病毒感染所需的宿主因子的隐性遗传筛查。
DOI：
10.1186/gb-2007-8-4-r48
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发表时间：
2007
期刊：
影响因子：
12.3
通讯作者：
Bradley, Allan
中科院分区：
文献类型：
--
作者：
Wang, Wei;Bradley, Allan
插入突变的Blm-/- ES细胞文库的隐性遗传筛选确定了逆转录病毒感染所需的宿主因子，并证实了mCat-1是ES细胞中的生态性小鼠白血病病毒受体。逆转录病毒感染所需的宿主因子是调控由逆转录病毒引起的疾病的潜在靶点。在逆转录病毒的生命周期中，许多细胞因子与病毒相互作用，并在感染中发挥重要作用。培养的胚胎干细胞（ES）易受逆转录病毒感染，因此提供了进入这一过程所需的所有基因的途径。为了确定参与逆转录病毒感染的宿主因子，我们设计并实施了一种方案，用于鉴定对逆转录病毒感染具有抗性的胚胎干细胞，并随后克隆突变基因。在blm缺陷胚胎干细胞中，采用全基因组插入诱变方法建立突变胚胎干细胞文库，并用携带阳性和阴性选择盒的重组逆转录病毒对文库进行高倍性重复感染筛选。然后使用严格的阴性选择来排除感染的胚胎干细胞。我们成功地恢复了5个能够抵抗逆转录病毒感染的独立胚胎干细胞克隆。对这些克隆的突变分析发现，mCat-1位点存在4个不同的纯合突变和1个复合杂合突变，证实了mCat-1是ES细胞中的生态嗜性小鼠白血病病毒受体。我们已经证明了这种隐性遗传方法用于鉴定胚胎干细胞中逆转录病毒感染所需的关键基因的可行性和可靠性；这种方法为恢复逆转录病毒感染所需的其他细胞因子提供了独特的机会。由此产生的插入突变的blm缺陷ES细胞文库也可能提供对其他类型病毒感染和复制所需的基本宿主细胞成分的访问。
A recessive genetic screen of an insertionally mutated Blm-/- ES cell library identifies host factors required for retroviral infection, and confirms that mCat-1 is the ecotropic murine leukaemia virus receptor in ES cells. Host factors required for retroviral infection are potential targets for the modulation of diseases caused by retroviruses. During the retroviral life cycle, numerous cellular factors interact with the virus and play an essential role in infection. Cultured embryonic stem (ES) cells are susceptible to retroviral infection, therefore providing access to all of the genes required for this process to take place. In order to identify the host factors involved in retroviral infection, we designed and implemented a scheme for identifying ES cells that are resistant to retroviral infection and subsequent cloning of the mutated gene. A library of mutant ES cells was established by genome-wide insertional mutagenesis in Blm-deficient ES cells, and a screen was performed by superinfection of the library at high multiplicity with a recombinant retrovirus carrying a positive and negative selection cassette. Stringent negative selection was then used to exclude the infected ES cells. We successfully recovered five independent clones of ES cells that are resistant to retroviral infection. Analysis of the mutations in these clones revealed four different homozygous and one compound heterozygous mutation in the mCat-1 locus, which confirms that mCat-1 is the ecotropic murine leukemia virus receptor in ES cells. We have demonstrated the feasibility and reliability of this recessive genetic approach to identifying critical genes required for retroviral infection in ES cells; the approach provides a unique opportunity to recover other cellular factors required for retroviral infection. The resulting insertionally mutated Blm-deficient ES cell library might also provide access to essential host cell components that are required for infection and replication for other types of virus.
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影响因子：
30.8
作者：
Luo, GB;Santoro, IM;Bradley, A
通讯作者：
Bradley, A
影响因子：
64.8
作者：
Deng, HK;Liu, R;Landau, NR
通讯作者：
Landau, NR
DOI：
10.1073/pnas.1633296100
发表时间：
2003-08-19
影响因子：
11.1
作者：
Hansen, J;Floss, T;Ruiz, P
通讯作者：
Ruiz, P
影响因子：
64.5
作者：
Ding, S;Wu, XH;Xu, T
通讯作者：
Xu, T
DOI：
10.1073/pnas.87.23.9202
发表时间：
1990-12-01
影响因子：
11.1
作者：
GREZ, M;AKGUN, E;OSTERTAG, W
通讯作者：
OSTERTAG, W