Part:BBa_K1470006
pMIG
pMIG is a retroviral transfer plasmid to produce viral vectors when it is transfected into a packaging cell line. With these viral vectors it is possible to efficiently create stable cell lines in just one week. pMIG contains two long terminal repeats (5'LTR and 3'LTR), which flank a psi packaging sequence and space for genes with a size of up to 8 kb. The 5'LTR serves as a strong promoter for the gene of interest (GOI), that can be inserted with the restriction sites EcoRI and PstI. This allows to use Biobricks of the RFC10 and RFC25 standard. The originally existing genes for the envelope, the capsid proteins and the polymerase and integrase are located in a packaging cell line (Phoenix eco). By transfecting pMIG to this cell line, functional viral vectors containing the gene of interest are produced. The viral particles are secreted into the cell culture supernatant and can be used to infect target cells. The viral particles are derived from a Murine Leukemia Virus and therefore can only insert the gene of interest into the genome of dividing cells. Read more about the viral vector
Origin, Standardization and Application
This plasmid was originally created by William Hahn and is available as plasmid 9044 at Addgene, we got his permission to submit pMIG as biobrick. It is a high copy plasmid and contains an Ampicillin resistance.
In order to provide this plasmid as a mammalian expression vector, we removed several EcoRI- and PstI-sites and an IRES. Afterwards we inserted a multiple cloning site, consisting of XhoI, EcoRI, NotI, SalI, PstI, HindIII and ClaI. However, it was not possible to delete XbaI sites in 3'LTR and 5'LTRs due to their nearly identical sequences. Nonetheless, any biobrick cut with EcoRI/PstI can be ligated into pMIG, hence providing future iGEM Teams a well functioning and reliable expression vector for mammalian cells.
Characterization
The viral vector uses the murine CAT-1 receptor (mCAT-1) as an entry site into the cell. Therefore, it can specifically transduce murine cells (e.g. NIH3T3), but not cell lines from other species (e.g. the human embryonic kidney cells (HEK-293T).
Figure 1: Infection of different cell lines with the viral vector derived from the murine leukemia virus.
Cells that were infected by viral particles express EGFP.
After transfection of human cells (HEK-293T) with the fusion protein mCAT-1-mCherry, viral vectors containing EGFP can also integrate into non-murine cells.
Figure 2: HEK-293T cells expressing mCAT-1-mCherry transduced with MuLV-GFP. (A) Overlay of all three channels, (B) DAPI, (C) EGFP and (D) mCAT-1-mCherry.
Different genes for fluorescent protein were inserted into pMIG. The produced viral vectors were used to infect murine NIH3T3 cells.
Figure 3: Murine NIH3T3 cells infected with our viral vector. The vector contained different fluorescent proteins: (left) EGFP, (middle) mKO, (right) mKate that were stably integrated into the genome
The viral vector can stably integrate genes. After the transduction we sorted positiv cells and passaged them 10 times. The amount of EGFP expressing cells were measured by FACS analysis.
Figure 4: Stable integration of EGFP into the genome of the target cells. (left) Histogram of fluorescence intensity at the first sorting step. (middle) Histogram of fluorescence intensity after sorting. (right) The fraction of fluorescent cells stays constant for 10 passages.
The workflow consists of the following steps: Transfection of the packaging cell line with pMIG containing the GOI, harvest of the viral vector in the cell culture supernatant and transduction of the target cells.
Figure 5: Workflow.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Plasmid lacks a prefix.
Plasmid lacks a suffix.
Illegal EcoRI site found at 4917
Illegal XbaI site found at 289
Illegal XbaI site found at 3711
Illegal PstI site found at 10 - 12INCOMPATIBLE WITH RFC[12]Plasmid lacks a prefix.
Plasmid lacks a suffix.
Illegal EcoRI site found at 4917
Illegal NheI site found at 99
Illegal NheI site found at 3521
Illegal PstI site found at 10
Illegal NotI site found at 4925 - 21INCOMPATIBLE WITH RFC[21]Plasmid lacks a prefix.
Plasmid lacks a suffix.
Illegal EcoRI site found at 4917
Illegal BglII site found at 4899
Illegal XhoI site found at 4905 - 23INCOMPATIBLE WITH RFC[23]Plasmid lacks a prefix.
Plasmid lacks a suffix.
Illegal EcoRI site found at 4917
Illegal XbaI site found at 289
Illegal XbaI site found at 3711
Illegal PstI site found at 10 - 25INCOMPATIBLE WITH RFC[25]Plasmid lacks a prefix.
Plasmid lacks a suffix.
Illegal EcoRI site found at 4917
Illegal XbaI site found at 289
Illegal XbaI site found at 3711
Illegal PstI site found at 10
Illegal NgoMIV site found at 3466
Illegal NgoMIV site found at 4123 - 1000INCOMPATIBLE WITH RFC[1000]Plasmid lacks a prefix.
Plasmid lacks a suffix.
Illegal BsaI site found at 520
Illegal BsaI site found at 541
Illegal BsaI site found at 3964
Illegal BsaI.rc site found at 1901
Illegal BsaI.rc site found at 4030
Illegal BsaI.rc site found at 4645
Illegal SapI site found at 818
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