CONSTRUCTION AND CHARACTERIZATION OF A HIGHLY COMPLEX RETROVIRAL LIBRARY FOR LINEAGE ANALYSIS
CONSTRUCTION AND CHARACTERIZATION OF A HIGHLY COMPLEX RETROVIRAL LIBRARY FOR LINEAGE ANALYSIS
复制标题
DOI：
10.1073/pnas.92.12.5704
复制
发表时间：
1995-06-06
影响因子：
11.1
通讯作者：
CEPKO, CL
中科院分区：
文献类型：
--
作者：
GOLDEN, JA;FIELDSBERRY, SC;CEPKO, CL
编码组织化学报告基因的无复制能力的逆转录病毒载体已被用于研究各种物种的直系关系。解释这种方法产生的数据的关键因素是鉴定同胞关系或克隆边界。使用其中每个成员都是独特的病毒文库可以大大促进这方面的分析。先前报道的含有约80个成员的鼠逆转录病毒文库证明了文库方法的实用性。然而，鼠文库中相对低数量的标签需要使用低感染率以在克隆分配中给出置信度。构建了一个复杂得多的文库，并进行了表征。构建CHAPOL文库，使得每个成员编码一个组织化学报告基因，并具有一个DNA标签，该标签来源于合成的具有> 1 × 10 - 6复杂度的简并寡核苷酸库（7）。在体外或体内感染细胞后测试该文库。通过PCR回收来自每个组织化学标记的细胞或细胞克隆的DNA标签，并进行测序以进行明确的鉴定。感染后已经鉴定了320个标签，到目前为止还没有发现来自一个以上独立感染的标签。因此，建议插入物的均匀分布，并且Monte Carlo分析预测> 10（4）个成员的复杂性。
Replication-incompetent retroviral vectors encoding histochemical reporter genes hale been used for studying lineal relationships in a variety of species, A crucial element in the interpretation of data generated by this method is the identification of sibling relationships, or conal boundaries, The use of a library of viruses in which each member is unique can greatly facilitate this aspect of the analysis, A previously reported murine retroviral library containing about 80 members demonstrated the utility of the library approach, However, the relatively low number of tags in the murine library necessitated using low infection rates in order to give confidence in clonal assignments, To obviate the need for low infection rates, a far more complex library was created and characterized, The CHAPOL library was constructed such that each member encodes a histochemical reporter gene and has a DNA tag derived from a degenerate oligonucleotide pool synthesized to have a complexity of > 1 x 10(7), The library was tested after infection of cells in vitro or in vivo. The DNA tag from each histochemically labeled cell or clone of cells was recovered by PCR and sequenced for unambiguous identification, Three hundred and twenty tags have been identified after infection, and so far no tag has been seen to result from more than one independent infection, Thus, an equal distribution of inserts is suggested, and Monte Carlo analysis predicts a complexity of > 10(4) members.