Tang Michelle C W, Jacobs Shelley A, Wong Lee H, Mann Jeffrey R
Theme of Genetic Disorders, Murdoch Childrens Research Institute, The Royal Children's Hospital, Victoria, 3052, Australia.
Genesis. 2013 Feb;51(2):142-6. doi: 10.1002/dvg.22366. Epub 2013 Feb 7.
Post-translational modifications to residues in core histones convey epigenetic information. Their function can be evaluated in amino acid substitution mutants, although to date this method has not been used in mice. To this end, we have evaluated gene targeting vectors designed for Cre recombinase-mediated conditional allelic replacement at the two unlinked genes encoding the histone variant H3.3. The conditional alleles consist of an uninterrupted wild-type H3.3 coding sequence upstream of a desired alternative or proxy coding sequence. The arrangement of two loxP sites allows Cre-mediated replacement of the wild-type coding sequence with the proxy. To demonstrate proof of principle, at each locus we replaced the wild-type coding sequence with a fluorescent reporter. This produced null alleles that will be useful to analyse the effects of H3.3 deficiency in development. Each targeting vector can readily be retrofitted with a proxy coding sequence encoding a modified H3.3 protein. Such vectors will allow for the conditional substitution of specific residues in order to dissect the roles of H3.3 post-translational modifications in development and disease.
核心组蛋白中残基的翻译后修饰传递表观遗传信息。它们的功能可以在氨基酸替代突变体中进行评估，尽管迄今为止该方法尚未在小鼠中使用。为此，我们评估了基因靶向载体，这些载体设计用于在编码组蛋白变体H3.3的两个不连锁基因处进行Cre重组酶介导的条件性等位基因替换。条件性等位基因由所需替代或替代编码序列上游的不间断野生型H3.3编码序列组成。两个loxP位点的排列允许Cre介导的野生型编码序列被替代物取代。为了证明原理，在每个位点我们用荧光报告基因取代了野生型编码序列。这产生了无效等位基因，将有助于分析H3.3缺陷在发育中的影响。每个靶向载体都可以很容易地用编码修饰H3.3蛋白的替代编码序列进行改造。这样的载体将允许对特定残基进行条件性替换，以便剖析H3.3翻译后修饰在发育和疾病中的作用。
