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plat-e: an efficient and stable system for transient packaging of retroviruses
Generated based on a 293T cell line. Plat-
E is superior to existing packaging cell lines in terms of efficiency, stability and safety.
New packaging structure for building platform
Ensure efficient and stable expression of viral structural proteins.
The traditional packaging structure uses MuLV-
LTR of gag-expression of viral structural gene
Pol and env while our packaging structure uses 100-
More effective folding than MuLV
LTR in 293T cells is combined with a Kozak consistent sequence upstream of the starting password, resulting in a viral structural protein in Plat-E cells.
In order to maintain the high titer of the reverse transcription virus under the pressure of drug selection, we inserted IRES (
(Internal nucleic acid entry site)
Sequence between genes encoding gag-
Pol or env, as well as genes that encode the optional markers in the packaging structure. Plat-
E cells can stably produce reverse transcription viruses with an average titer of 1 × 10. 7/ml for at least 4 months.
In addition, since we only use the coding sequence of the virus structure gene to avoid including unnecessary reverse transcription virus sequences in the packaging structure, the possibility of producing replicable reverse transcription viruses (RCR)
It can actually be ruled out by restructuring.
Reverse transcription viral vectors and packaged cells are important tools for gene transfer applications.
Introduction of reverse transcription viral vectors containing genes of interest into suitable packaged cells can produce infectious reverse transcription viruses, which can infect target cells and stably transfer interested genes
In the traditional strategy, a stable high-producer of reverse transcription viral vectors containing genes of interest was established by converting the reverse transcription viral constructs into NIH3T3-
Packaging-based cells such as PA317 and 2-3 months are often required to acquire high producers.
Pear has developed a unique packaging system that can obtain high-priced reverse transcription viruses within 3 days through short-term transfer.
In Bosc23 cells, the expression of viral structural genes is driven by MuLV LTR.
Bosc23 cells and pMX-for transient transfer-
The Neo vector produces 1-3 × 10/ml virus, which is evaluated based on the number of neaccording-resistant colonies of infected NIH3T3 cells (data not shown).
Since Bosc23 cells carry large T antigens, we tried to increase the titer of the reverse transcription virus by introducing SV40 sources into the pMX vector to amplify the vector.
However, it turns out that this is not feasible (data not shown)
This suggests that the limiting factor is the level of expression of viral structural proteins in packaged cells.
Bosc23 was obtained by co-transfection of the encoded plasmid with the encoded Bosc23
The resistance gene is encoded with a vector encoding another selection gene GPT (
Guanidin phosphate transfer enzyme)
One by one
Therefore, the expression of selectable markers cannot be guaranteed or the expression of genes, which may be that cells produce high
Titer reverse transcription virus
Similar packaging cell line Phoenix-
E was also developed. In Phoenix-
In E cells, the vectors of the coding and gene were co-transferred with the selection marker, which did not guarantee the stable expression of the gene in the selection of drug tide and Baibai toxin, respectively.
Phoenix has several improvements
Compared with Bosc23 cells, E cells.
First of all, much stronger than MuLV-promoter for hiv and MV
LTR expression and genes in 293T cells were used, respectively.
Second, the internal nucleic acid entry site (IRES)
Sequences are used for simultaneous expression and cell surface labeling of CD8, which enables sequencing of high expression of genes.
However, in order to maintain the level of gene expression, one needs to classify cells from time to time.
In order to design a packaged cell line capable of stabilizing the production of high-titer reverse transcription viruses, we used facs-looking for a strong promoter to drive the expression of viral structural proteins in 293T cellsGAL assay.
In the 7 promoters detected, the ef1 α and PCR promoters induced ().
The activities promoted by these sponsors are 100-
It is twice higher than the LTR used in Bosc23 cells, even exceeding the SV40 and sr α promoters, which enables in 293T cells expressing SV40 large T antigens
Because we believe that the promoter of the housekeeping gene is more suitable for driving stable gene expression in mammalian cells than the virus promoter, we use the ef1 α promoter to express virus structural proteins in 293T cells ().
In addition, in the packaging structure described here, IRES inserted between the or gene and the selection marker.
Therefore, the expression of the selected marker is a direct reflection of or expression in the same cell.
PEnv-packaging structureIRES-puro and pGag-pol-IRES-
Bs constructed as described above were then transformed into 293T cells in sequence and 50 subclones resistant to both puromycin and blasticidin were isolated.
In 50 subclones, 1 clone is named Platinum-E (Plat-E)
A reverse transcription virus with the highest infection efficiency was produced and used for further analysis.
When tested on NIH3T3 cells, the titer of the reverse transcription virus is about 1 × 10/ml, using Plat-
E cells with pMX-transferlacZ (data not shown).
Next, we compare the earlier paragraphs of Plat.
Bosc23 cells and E cells of Phoenix-
E cells about long-
Long-term stable production
Reverse transcription virus with instantaneous titer ().
The culture conditions of the three packaged cell lines are as follows: Bosc23 cells grow in broth containing 10% fetal bovine serum containing GPT selection reagents indicated by the manufacturer (
Professional Media, Lavalette, New Jersey, United States of America). Phoenix-
E-cells were isolated by flow cell to express CD8 and preserved together with 10% fetal bovine serum containing tide (300u2009μg/ml)
And broken toxins (1u2009μg/ml)
After 1 week, the cells were transferred into a broth containing 10% fetal bovine serum free of tide and white diphtheria toxin. Plat-
E cells are always kept in broth containing 10% fetal bovine serum (10u2009μg/ml)and puromycin (1u2009μg/ml).
On the one hand, the infection efficiency of the reverse transcription virus produced by Bosc23 decreased within 3 months, and the infection efficiency of the reverse transcription virus produced by Phoenix decreased.
E cells are also reduced in time ().
Platform, on the other hand-
E a reverse transcription virus with infection efficiency greater than 75% was produced under drug selection pressure, with a titer of about 1 × 10/ml for at least 4 months.
Comparison of expression levels of Plat and mRNA
E, Bosc23 and Phoenix-
E-packed cell lines, Northern blot analysis using cells cultured for 3 weeks.
And the expression level of mRNA is 4-fold and 10-
High folding on the platform
E cells than other packaged cell lines ().
RT activity in cell lysate was also analyzed. Plat-
Compared to Bosc23 and Phoenix, E produces at least twice the RT activityE cells ().
In addition, the expression level of protein is much higher than that of Bosc23 and Phoenix-E ()
When evaluated by antibody staining against env gene products.
Since the reverse transcription virus structure genes are encoded on two different vectors, three recombinant events are required to produce replicable reverse transcription viruses (RCR).
In addition, the probability of recombination was minimized by using only the coding sequences of genes isolated from the MuLV genome in the packaging structure using PCR.
In fact, the production of RCR was tested through XC patch analysis and from Plat-
PMX-E cells after transfectionGFP.
As for the positive control, MoMuLV-
Infected C3H2K cells (
A gift from Dr. Xingye)
Used after continuous dilution, wild-
The MoMuLV type produced by C3H2K cells is estimated to be 1 × 10/ml.
In conclusion, we are reporting here a stable platform for reverse transcription virus packaging cell lines
There are several advantages over the existing packaging cell lines.
First, the ef1 α promoter in the packaging structure binds the consistent sequence of Kozak, allowing the production of a reverse transcription virus with a titer of 1 × 10/ml.
Second, the packaging structure uses a double-Shunlian vector carrying the IRES sequence to ensure the stable expression of the virus structure gene under drug selection pressure, this makes it possible to maintain the titer of the reverse transcription virus from the platform.
E cells were obtained by simply cultivating cells in the presence of the selected drug.
Finally, in order to reduce the possibility of producing RCR, the minimum virus sequence was used in the packaging structure. Thus, Plat-
E cells can produce auxiliary cells stably
Free reverse transcription virus with high titer for a long time.
Reverse transcription virus using Plat
E cells, we can effectively transfer genes to many different cells, including primary cultured cells such as T cells and mast cells (data not shown).
Recently, it has been reported that by importing the coding region of the early genes of the multi-tumor virus into packaged cell lines, the recombinant reverse transcription virus drops produced by these cell lines are 10-100 times higher than those produced by the mother cell lines.
Import the early stage of multi-tumor virus into the platform
E may lead to more efficient production of high-titer reverse transcription viruses. Plat-
E is an ecological packaging cell line, its ecological packaging cell line Plat-
Cell lines should prove useful in human gene therapy.