Passenger Gene Mutations: Unwanted Guests in Genetically Modified Mice.
Passenger Gene Mutations: Unwanted Guests in Genetically Modified Mice.
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DOI：
10.1002/jbmr.2772
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发表时间：
2016-02
期刊：
影响因子：
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通讯作者：
Rosen CJ
中科院分区：
文献类型：
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作者：
Ackert-Bicknell CL;Rosen CJ
携带基因突变的小鼠经常被用来确定特定基因或突变在骨骼生理学和病理学中的作用。最常使用的三种主要模型是工程突变(基因敲除、转基因、敲打等)、化学诱导突变和自发突变。这些模型很大程度上是在近交系小鼠身上制造或重新发现的(取决于类型)，而不是近交系小鼠。然而，由于几个原因，通常有必要改变感兴趣等位基因的背景菌株。最常见的理由是，由于菌株的某些特性，如繁殖性能，背景菌株是不受欢迎的。其他原因包括有必要将两种突变相互比较的实验，或者研究在一只动物身上存在两个等位基因的动物。在后一种情况下，有必要在相同的遗传背景上有突变(S)，以将感兴趣的等位基因(S)的影响与任何两个背景之间的遗传差异所造成的差异分开。尽管现在有更多关于用于制造转基因的胚胎干细胞(ES)的菌株背景的选择，例如通过同源重组进行的传统敲除，但情况并不总是如此。因此，许多转基因小鼠是在129或FVB背景下制造的，因为在这些背景下制造转基因小鼠相对容易。(1，2)然而，129小鼠可能很难培育，(3)FVB小鼠作为敲除小鼠是不受欢迎的，因为这种背景不太常见，使得两个突变体之间的正面比较变得困难。自发突变是罕见的随机事件，可能在任何时间、任何背景下出现。因此，习惯上使用回交方法将突变或转基因从原始背景转移到“标准”C57BL/6(B6)菌株上(图1)。简而言之，突变的小鼠(供体品系)被培育成B6小鼠，而携带目标突变的后代又被培育成B6小鼠。这些后代进行了基因分型，携带者再次繁殖到B6小鼠，以此类推。这一过程理想地重复10代，因为从统计上看，99.8%的菌株现在是类似B6的，来自供体129菌株的污染只有0.2%。(4)这被称为同源菌株，这种同源菌株的背景历史通常反映在菌株名称中。例如，商业上可获得的Sox9等位基因的官方菌株名称是B6。129S7-Sox9tm2Crm/J(杰克逊实验室；https://www.贾克斯。Org/)，反映了该菌株是用129SvEv株(5)的ES细胞制成的，并且通过至少9次回交将开花等位基因转移到B6背景。不幸的是，关于供体菌株0.2%的污染与感兴趣的转基因或等位基因的关系，通常很少或根本没有讨论。这第一点是文章的题目《乘客突变困惑》
Mice carrying genetic mutations are used with great frequency to determine the role of a specific gene or mutation in skeletal physiology and pathology. The three main models most frequently employed are engineered mutants (knockouts, transgenics, knockins, etc.), chemically induced mutations, and spontaneously occurring mutations. These models are largely made or de novo discovered (depending on the type) in inbred strains of mice as opposed to outbred lines. However, for several reasons, it is often necessary to change the background strain of an allele of interest. The most common rationale is that the background strain is undesirable due to some characteristic of the strain, such as breeding performance. Other reasons include experiments where it is necessary to compare two mutations to each other, or to study animals with two alleles present in one animal. In these latter cases, it is necessary to have the mutation (s) on the same genetic background to separate the effects of the allele (s) of interest from the differences caused by the genetic dissimilarities between any two backgrounds. Although there are now more choices available with regard to the strain background of the embryonic stem (ES) cells used to make a transgenic such as a traditional knockout made via homologous recombination, this was not always the case. As a result, many transgenic mice were made on a 129 or FVB background, because it is comparatively easy to make transgenic mice on these backgrounds.(1, 2) However, 129 mice can be difficult to breed,(3) and FVB mice are undesirable as knockouts because this background is less common, making head-to-head comparisons between two mutants difficult. Spontaneous mutations are rare random events and could appear at any time, on any background. Thus, it is customary to move a mutation or transgene from the original background onto a “standard” C57BL/6 (B6) strain using a backcross approach (Fig. 1). In short, the mutant mouse (donor strain) is bred to a B6 mouse and the offspring carrying the mutation of interest are bred again to B6 mice. These offspring are genotyped and the carriers are bred again to B6 mice, and so on. This process is ideally repeated for 10 generations, because statistically $99.8% of the strain is now B6-like and there is only contamination of 0.2% from the donor 129 strain.(4) This is called a congenic strain and the background history of such a congenic is often reflected in the strain name. For example, the official strain name for the commercially available Sox9 floxed allele is B6. 129S7-Sox9tm2Crm/J (The Jackson Laboratory; https://www. jax. org/), reflecting that this strain was made using ES cells from a 129SvEv strain (5) and that the floxed allele was moved to a B6 background by backcrossing at least nine times. It is unfortunate that there is often little or no discussion as to where this 0.2% contamination by the donor strain may be found relative to the transgene or allele of interest. This first point is the topic of the article Passenger Mutations Confound