Related Experiment Video
Updated: Nov 10, 2025
Flow Cytometry to Estimate Leukemia Stem Cells in Primary Acute Myeloid Leukemia and in Patient-derived-xenografts, at Diagnosis and Follow Up
Published on: March 26, 2018
[Construction and Identification of Leukemia Cell Line Stably Expressing CD123 and CLL-1]
Xiang-Yu Wang1, Guo-Qiang Lin1, Lei Yu2
1Department of Hematology, Huai'an Hospital Affiliated to Xuzhou Medical College and Huai'an Second People's Hospital, Huai'an 223002, Jiangsu Province, China.
Objective:
To construct an acute myeloid leukemia cell line stably expressing CD123-CLL1 so as to provide an "in vitro" model for studying the role of CD123 and CLL-1 in leukemia and the treatment targeting CD123 and CLL-1.
Methods:
The recombinant plasmid of lentivirus was constructed by synthesizing CD123 and CLL-1 sequences and PCR homologous recombination. The lentivirus vector was packaged by three-plasmid packaging system. After collecting the supernatant of lentivirus, the virus titer was determined by quantitative PCR. K562 leukemia cells were collected and transtected with virus supernatant. Leukemia cell line stably expressing the target gene were screened by purinomycin. The expression levels of CD123 and CLL-1 were detected by RT-PCR and flow cytometry.
Results:
The lentiviral vector was successfully constructed, and identified by agarose gel electrophoresis and gene sequencing, then the virus titer of the supernatant was up to 5.81×108 after quantitative PCR assay. The K562 leukemia cell line obtained positive expression cells after being infected by puromycin. The high expression of CD123 and CLL-1 was confirmed by RT-PCR, while the significantly high expression of CD123 and CLL-1 was confirmed by flow cytometry.
Conclusion:
Lentiviral vector expressing CD123-CLL1 has been successfully constructed, and K562 leukemia cell line stably expressing CD123 and CLL-1 has been successfully obtained.
