Dolnikov A, Millington M, Beruter E, Shounan Y, Symonds G
The School of Physiology and Pharmacology, The University of New South Wales, Randwick, NSW, Australia.
J Hematother Stem Cell Res. 2000 Oct;9(5):659-72. doi: 10.1089/15258160050196704.
We have used a simple, single-gene retrovirus carrying the Escherichia coli beta-galactosidase reporter gene (lacZ), termed LlacZ. This virus was found to infect immortalized myeloid and lymphoid precursor/leukemic cell lines efficiently as well as primary murine bone marrow clonogenic progenitors, without apparent modulation of growth or phenotype. Following infection of bone marrow cells, a significant proportion of progenitors--36% of lineage-negative cells with low levels of c-kit expression (lin-/c-kit(lo)) known to be enriched with pluripotent hemopoietic stem cells, and 19% of Sca1-positive cells known to be enriched with transplantable cells with lymphomyeloid-reconstituting ability--were shown to express lacZ. Use of an LlacZ-infected population of post 5-fluorouracil bone marrow cells to reconstitute lethally irradiated mice demonstrated the presence of lacZ-expressing cells in the spleen at day 12 post-transplantation with provirus detected in individual spleen colonies (CFU-S). In the long term (3-6 months following transplantation), lacZ expression was detected in hematopoietic tissues of all recipient mice. The use of two-color in situ and flow cytometry analysis combined with lineage-specific antibodies showed lacZ expression in both myeloid and lymphoid cells in spleen and bone marrow. In addition, lacZ-expressing cells were detected in secondary recipient mice injected with bone marrow cells derived from primary LlacZ recipients. Overall, these data show the efficacy of a single gene vector for stem cell transduction, the utility of beta-galactosidase as a single cell marker for stem cell transduction and reconstitution ability, and the need for protocol optimization to see high-level multilineage gene expression.
我们使用了一种携带大肠杆菌β-半乳糖苷酶报告基因(lacZ)的简单单基因逆转录病毒,称为LlacZ。发现这种病毒能有效感染永生化的髓系和淋巴系前体/白血病细胞系以及原代小鼠骨髓克隆形成祖细胞,且对细胞生长或表型无明显调节作用。骨髓细胞感染后,相当一部分祖细胞——36%的c-kit低表达(lin-/c-kit(lo))的谱系阴性细胞(已知富含多能造血干细胞)和19%的Sca1阳性细胞(已知富含具有淋巴细胞系重建能力的可移植细胞)——被证明表达lacZ。用LlacZ感染的5-氟尿嘧啶处理后的骨髓细胞群体重建经致死性照射的小鼠,结果表明移植后第12天脾脏中存在表达lacZ的细胞,且在单个脾脏集落(CFU-S)中检测到前病毒。在长期(移植后3 - 6个月),所有受体小鼠的造血组织中均检测到lacZ表达。使用双色原位和流式细胞术分析并结合谱系特异性抗体显示,脾脏和骨髓中的髓系和淋巴系细胞中均有lacZ表达。此外,在注射来自原发性LlacZ受体的骨髓细胞的二次受体小鼠中也检测到了表达lacZ的细胞。总体而言,这些数据表明单基因载体用于干细胞转导的有效性、β-半乳糖苷酶作为干细胞转导和重建能力的单细胞标记的实用性,以及为实现高水平多谱系基因表达而进行方案优化的必要性。