A retroviral strategy that efficiently creates chromosomal deletions in mammalian cells. Mus musculus

Background We have developed and validated a set of complementary retroviruses that creates a wide range of nested chromosomal deletions. When applied to mouse ES cells, this retrovirus-based method generated deletions ranging from 6 kb to 23 Mb (average 2.9 Mb), with an efficiency of 64% for drugs-selected clones. Importantly, several of engineered ES cell clones, mostly those with large deletions, showed major alteration in cell fate. The set of complementary replication-defective retroviruses exploited the Cre-loxP recombination system and reconstitution of a functional neomycin cassette for selection of recombination events. The first loxP sequenced was delivered using the anchor virus A1. The integration was selected on puromycin. ES cell clones containing one copy of the virus A1 were isolated and expanded (primary clones). A second loxP was introduced by retroviral gene transfer using the saturation virus S1, selected on hygromycin (secondary clones). The transient expression of the recombinase Cre in secondary clones allowed the recombination between the loxP sites and the functional reconstitution of a split neomycin expression cassette. Neomycin resistant clones (tertiary clones) were isolated and expanded. Chromosomal deletions are expected to have occurred in neomycin resistant clones that have lost both puromycin and hygromycin resistance genes. In addition, other chromosomal rearrangements (e.g., inversions, translocations, etc.) are achievable using this system. Aim Inverse-PCR, array-based comparative genomic hybridization (aCGH) and spectral karyotyping were employed to confirm deletions (or other Cre-induced rearrangements) in several tertiary clones and to assess the genomic integrity of the ES cells altered. Conclusions aCGH conclusions are summarized together with complementary results from inverse-PCR and spectral karyotyping in 3 tables: Table 1: Summary of the virus A1 integration Table 2: Characteristics of independent deletions confirmed by IPCR-aCGH Table 3: Confirmed or suspected recombination events in trans Notes for Table 2: Mapping and deletion analyses were done using the UCSC Genome Browser (NCBI mouse Build 33). {a} Tertiary clones are labeled according to their family number (same integration of virus A1), followed by a specific id number. If more than one clone presented a redundant rearrangement within the same group infected with virus S1, only one is reported for clarity. {b} Anomaly that was not present in the primary clone from which the tertiary clone was derived, as determined by aCGH or SKY. ( -), no anomaly; (+), additional anomaly. {c} The deletion is not observed, in agreement with the resolution of aCGH. {d} Normal except for the loss of chromosome Y. {e} Amplification of chromosome 1. {f} Amplification of chromosome 8. {g} Many chromosomes were lost according to SKY. {h} Amplification on chromosome 14. Id, identification; kb, kilobase pairs; no., number; aCGH, array-based comparative genomic hybridization; SKY, spectral karyotyping; I-PCR, inverse-PCR; n.d., not determined. Keywords: comparative genomic hybridization Overall design: aCGH (n=30) were conducted between our mouse ES cell samples and a control normal mouse genomic DNA. Selected tertiary clones (n= 20) were compared to their corresponding primary clones (n=9), in order to find anomalies that was not present in the primary clones from which they were derived. Primary clones were compared together and to the unmodified R1 ES cells (n=1) to assess the genomic integrity.

研究背景
我们开发并验证了一套互补型逆转录病毒（retrovirus）系统，可诱导产生多种嵌套式染色体缺失。将该基于逆转录病毒的方法应用于小鼠胚胎干细胞（mouse ES cells）时，可生成长度介于6 kb至23 Mb之间（平均2.9 Mb）的染色体缺失，经药物筛选的克隆的重组效率达64%。值得注意的是，部分工程化胚胎干细胞克隆（尤其是携带大片段缺失的克隆）的细胞命运发生了显著改变。
这套互补型复制缺陷型逆转录病毒系统借助Cre-loxP重组系统（Cre-loxP recombination system）实现功能，并通过重构功能性新霉素表达盒（neomycin cassette）来筛选重组事件。首个loxP位点通过锚定病毒A1（anchor virus A1）递送至基因组，其整合事件通过嘌呤霉素（puromycin）筛选得以富集。携带1份病毒A1拷贝的胚胎干细胞克隆被分离并扩增，即原代克隆（primary clones）。
随后通过饱和病毒S1（saturation virus S1）进行逆转录病毒基因转导，引入第二个loxP位点，并通过潮霉素（hygromycin）筛选获得二次克隆（secondary clones）。在二次克隆中瞬时表达重组酶Cre，可介导两个loxP位点间的重组，并重构分裂型新霉素表达盒使其恢复功能。随后分离并扩增新霉素抗性克隆，即三次克隆（tertiary clones）。
在同时丢失嘌呤霉素与潮霉素抗性基因的新霉素抗性克隆中，理论上已发生染色体缺失。此外，该系统还可用于诱导其他染色体重排事件（如倒位、易位等）。

研究目的
本研究采用反向PCR（inverse-PCR）、阵列比较基因组杂交（array-based comparative genomic hybridization, aCGH）以及光谱核型分析（spectral karyotyping, SKY）技术，对多个三次克隆中的染色体缺失（或其他Cre诱导的染色体重排）进行验证，并评估发生遗传改变的胚胎干细胞的基因组完整性。

研究结论
阵列比较基因组杂交（aCGH）结果结合反向PCR与光谱核型分析的补充结果，汇总于以下3张表格中：
表1：病毒A1整合事件汇总
表2：经反向PCR-aCGH验证的独立缺失事件特征
表3：经确认或疑似的反式重组事件

表2注释：
{a} 三次克隆以其家族编号（对应病毒A1的相同整合位点）加特定ID编号命名。若同一病毒S1感染组内存在多个冗余重排的克隆，为简洁起见仅报告1例。
{b} 指该异常未出现在生成该三次克隆的原代克隆中，经aCGH或SKY验证。（-）表示无异常；（+）表示存在额外异常。
{c} 未观察到缺失，与aCGH的分辨率限制相符。
{d} 除Y染色体丢失外无其他异常。
{e} 1号染色体扩增。
{f} 8号染色体扩增。
{g} 经SKY检测多条染色体丢失。
{h} 14号染色体区域扩增。
术语注释：Id（identification，识别编号）；kb（kilobase pairs，千碱基对）；no.（number，数量）；aCGH（array-based comparative genomic hybridization，阵列比较基因组杂交）；SKY（spectral karyotyping，光谱核型分析）；I-PCR（inverse-PCR，反向PCR）；n.d.（not determined，未测定）。
关键词：比较基因组杂交

实验整体设计
以正常小鼠基因组DNA为对照，对30份小鼠胚胎干细胞样本开展阵列比较基因组杂交（aCGH）检测。选取20份三次克隆样本，分别与其对应的9份原代克隆样本进行比对，以筛选出其来源原代克隆中不存在的异常。同时将各原代克隆之间以及与未修饰的R1胚胎干细胞（n=1）进行比对，以评估基因组完整性。