| Successful modification | The parasite was generated by the genetic modification |
| The mutant contains the following genetic modification(s) |
Introduction of a transgene,
Introduction of a transgene
|
| Reference (PubMed-PMID number) |
Reference 1 (PMID number) : 27102897
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| MR4 number |
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| Parent parasite used to introduce the genetic modification |
| Rodent Malaria Parasite | P. berghei |
| Parent strain/line | P. berghei ANKA |
| Name parent line/clone |
RMgm-928
|
| Other information parent line | A P. berghei reference line expressing mCherry under the control of the P. berghei hsp70 promoter. The mutant does not contain a drug-selectable marker. The mCherry expression cassette has been introduced into the silent p23p locus |
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| The mutant parasite was generated by |
| Name PI/Researcher | De Niz M; Heussler VT |
| Name Group/Department | Institute of Cell Biology |
| Name Institute | University of Bern |
| City | Bern |
| Country | Switzerland |
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| Name of the mutant parasite |
| RMgm number | RMgm-1441 |
| Principal name | mCherry(Hsp70)NLuc(ef1α) |
| Alternative name | PbNLuc |
| Standardized name | |
| Is the mutant parasite cloned after genetic modification | Yes |
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| Phenotype |
| Asexual blood stage | Expression of NanoLuc in all life cycle stages (in vitro) |
| Gametocyte/Gamete | Expression of NanoLuc in all life cycle stages (in vitro) |
| Fertilization and ookinete | Expression of NanoLuc in all life cycle stages (in vitro) |
| Oocyst | Expression of NanoLuc in all life cycle stages (in vitro) |
| Sporozoite | Expression of NanoLuc in all life cycle stages (in vitro) |
| Liver stage | Expression of NanoLuc in all life cycle stages (in vitro) |
| Additional remarks phenotype | Mutant/mutation
The mutant expresses NanoLuc under the control of the constitutive eef1a promoter. The expression cassette is introduced by single cross-over integration into the (silent) c/d-small subunit rrna gene unit. In addition, it expresses mCherry under the control of the constitutive hsp70 promoter. This expression cassette is introduced by double cross-over integration into the silent 230p locus.
Protein (function)
NanoLuc is a 19 kDa enzyme, synthetically generated following purification of the natural enzyme from the deep sea shrimp Oplophorus gracilorostris. NanoLuc possesses 150-fold brighter luminescent signal than firefly or Renilla. Aside from its high level of luminescence, NanoLuc is able to sustain signal intensity with a relatively long half-life of 2 h, and generates low background signal. In addition to optimal activity of the enzyme, suitable substrates were investigated to further maximize protein stability and light output. The coelenterazine analogue furimazine, in combination with NanoLuc, was found to produce the brightest output, and enhanced stability. NanoLuc has been used in dual or multiplex assays in combination with Gaussia or firefly luciferases.
Phenotype
Expression of NanoLuc in all life cycle stages (in vitro)
Additional information
Compared to other luciferases, no (less) suitable substrates are available for in vivo imaging in mice.
Although the spectral profile difference of NanoLuc compared to the other luciferases is an advantage in vitro, the use of blue-shifted luciferases in vivo poses an challenge, as short wavelengths do not readily penetrate mammalian tissues. Although other work has used NanoLuc for characterizing events at skin level, imaging of deep organs in living mice, including the liver, lungs, spleen, and brain was attempted in this study and was found to be unsuccessful.
Other mutants |
*RMgm-1441
