Establishment of the methodology for quantifying lentiviral vector transcriptional read-through rate.
- Author:
Jiaping HE
1
;
Yudan FANG
;
Fan ZHANG
;
Fengqiang SUN
;
Juan WANG
;
Jingzhi ZHANG
Author Information
1. Shanghai Institute of Medical Genetics, Children's Hospital of Shanghai, Children's Hospital Affiliated to Shanghai Jiao Tong University, Shanghai 200040, China.
- Publication Type:Journal Article
- MeSH:
Flow Cytometry;
Genetic Therapy;
Genetic Vectors;
Green Fluorescent Proteins;
biosynthesis;
HEK293 Cells;
Humans;
Lentivirus;
Transfection
- From:
Chinese Journal of Biotechnology
2013;29(7):1006-1015
- CountryChina
- Language:Chinese
-
Abstract:
As an effective vehicle for bio-research and for gene therapy, Lentiviral Vector (LV) has been drawn large attention in recent years. However, transcriptional read-through limits its application. In order to understand the extend of LV read-through in chromosome, a reliable method to assess transcriptional read-through rate is needed. Here, we report the method as follows: 293T cells were transfected with the lentiviral transfer vectors which borne with two LTRs at its two ends in order to mimic the state of "proviral vectors" in chromosome. Using the primers specific for 3'U5 and 3'U3, read-through and total transcripts were reverse transcribed, respectively. These two cDNAs were quantified by realtime PCR using the primers and probe specific for 5'end of 3'U3. Read-through rate was then calculated by the division of the two. Meanwhile, read-through product of green fluorescence protein was also analyzed by Fluorescence Activated Cell Sorter. They both reciprocally proved the principal and confirmed that self-inactivated LV appeared higher read-through rate than the wild type one. The method described in this article, therefore, provides a useful technique to study how to reduce read-through rate, and improve the bio-safety of LV.
