Summary

RMgm-590
Malaria parasiteP. berghei
Genotype
Transgene
Transgene not Plasmodium: HIV-1 gag
Promoter: Gene model: PBANKA_1133300; Gene model (P.falciparum): PF3D7_1357100; Gene product: elongation factor 1-alpha (eef1a)
3'UTR: Gene model: PBANKA_0719300; Gene product: bifunctional dihydrofolate reductase-thymidylate synthase, putative (dhfr/ts)
Phenotype Asexual bloodstage;
Last modified: 23 October 2010, 21:14
  *RMgm-590
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Introduction of a transgene
Reference (PubMed-PMID number) Reference 1 (PMID number) : 20933565
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei NK65
Name parent line/clone Not applicable
Other information parent line
The mutant parasite was generated by
Name PI/ResearcherB. Jiang; X. Chen
Name Group/DepartmentCenter for Infection and Immunity
Name InstituteGuangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences
CityGuangdong
CountryChina
Name of the mutant parasite
RMgm numberRMgm-590
Principal namePbGAG
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageWestern blot analysis showed that the mutant expressed a high level of the full-length 55kDa Gag protein using an anti-Gag antibody
Gametocyte/GameteNot tested
Fertilization and ookineteNot tested
OocystNot tested
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant expresses the transgene HIV1-GAG.  The gag gene was amplified from the genomic DNA of an HIV-1 infected donor from Henan Province of China (an HIV epidemic area) and was identified as of subtype B through sequence analysis, using the primer5'Gag (BamHI), 5'-CGGGATCCATGGGTGCGAGAGCGTC-3', and 3'Gag (BamHI), 5'-CGGGATCCTTATTGTGACGAGGGGTCG-3'. Although the DNA vector used to transfect the parasites contains targeting sequences for stably integration into the p230 locus, the DNA vector has been introduced and maintained as an episome. The gag gene is under the control of the constitutive eef1a promoter

Protein (function)
The Gag proteins of HIV-1, like those of other retroviruses, are necessary and sufficient for the assembly of virus-like particles. The roles played by HIV-1 Gag proteins during the life cycle are numerous and complex, involving not only assembly but also virion maturation after particle release and early post-entry steps in virus replication. As the individual Gag domains carry out their diverse functions, they must engage in interactions with themselves, other Gag proteins, other viral proteins, lipid, nucleic acid (DNA and RNA), and host cell proteins.
HIV-1Gag-specific responses have been shown to be associated with immune protection as demonstrated by decreases in viremia and viral load during chronic infection in patients. Monkeys vaccinated with the rAd26/rAd5 vector encoding SIV Gag showed lower viral loads as well as decreased AIDS-related mortality as compared with control animals after SIVmac251 challenge

Phenotype
Western blot analysis showed that the blood stages of the mutant expressed a high level of the full-length 55kDa Gag protein using an anti-Gag antibody.

Additional information
It was observed that the HIV-1 Gag polyprotein was proteolytically processed in P. berghei.

BALB/c mice mice vaccinated with PbGAG were significantly protected from challenge with vaccinia virus-gag (VV-gag) with an average 30-fold reduction in titer (P < 0.05). In addition, mice immunized with PbGAG developed Plasmodium specific immune responses and the immunized animals were protected from challenges with blood-stage P. berghei NK65 and Plasmodium yoelii 17XL.

The following immunization protocol was used:
Mice were infected intraperitoneally (i.p.) with 107 blood stages parasites of PbGAG.3-5 days after the immunization,  the parasite densities in blood in the mice reached 1–3%. In order to allow long-term infections in mice with transgenic P. berghei, the parasitemias were fairly controlled by adding sulfadiazine to the drinking water (10mg/l) for 2–4 consecutive days(whenever parasite densities were at the levels of 1–3%). After the drug administration for the duration mentioned above, the parasite densities decreased to barely detectable levels. Removing sulfadiazine from the drinking water resulted in a rebound of parasitemias. When the parasite densities reached the levels of 1–3%, the sulfadiazine was added again for a same duration. This procedure was repeated for two to three times. After that,the mice were immune, and withdrawing sulfadiazine no longer resulted in a rise in parasitemias. The mice were intragastrically administered with pyrimethamine (25 mg/kg)daily from day 1 to day 20 to maintain the plasmids in the transgenic parasites. During this time, folinic acid (400 µg/kg) was i.p. administered once a week to counter act bone marrow suppression induced by pyrimethamine treatment. All of the mice were intragastrically administered with chloroquine (80mg/kg) from days 21 to 24 to kill the remaining parasites to terminate the immunization.

Other mutants


  Transgene: Mutant parasite expressing a transgene
Type and details of transgene
Is the transgene Plasmodium derived Transgene: not Plasmodium
Transgene nameHIV-1 gag
Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/constructCircular plasmid
PlasmoGEM (Sanger) construct/vector usedNo
Modified PlasmoGEM construct/vector usedNo
Plasmid/construct map
Plasmid/construct sequence
Restriction sites to linearize plasmid
Selectable marker used to select the mutant parasitetgdhfr
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationThe gag gene was amplified from the genomic DNA of an HIV-1 infected donor from Henan Province of China (an HIV epidemic area) and was identified as of subtype B through sequence analysis, using the primer5'Gag (BamHI), 5'-CGGGATCCATGGGTGCGAGAGCGTC-3', and 3'Gag (BamHI), 5'-CGGGATCCTTATTGTGACGAGGGGTCG-3'.

Although the DNA vector used to transfect the parasites contains targeting sequences for stably integration into the p230 locus, the DNA vector has been introduced and maintained as an episome. The gag gene is under the control of the constitutive eef1a promoter
Additional remarks selection procedure
Other details transgene
Promoter
Gene Model of Parasite PBANKA_1133300
Gene Model P. falciparum ortholog PF3D7_1357100
Gene productelongation factor 1-alpha
Gene product: Alternative nameeef1a
Primer information details of the primers used for amplification of the promoter sequence  Click to view information
Primer information details of the primers used for amplification of the promoter sequence  Click to hide information
Sequence Primer 1
Additional information primer 1
Sequence Primer 2
Additional information primer 2
3'-UTR
Gene Model of Parasite PBANKA_0719300
Gene productbifunctional dihydrofolate reductase-thymidylate synthase, putative
Gene product: Alternative namedhfr/ts
Primer information details of the primers used for amplification the 3'-UTR sequences  Click to view information
Primer information details of the primers used for amplification the 3'-UTR sequences  Click to hide information
Sequence Primer 1
Additional information primer 1
Sequence Primer 2
Additional information primer 2
Insertion/Replacement locus
Replacement / InsertionNot available
Gene Model of Parasite Not available
Gene productNot available
Gene product: Alternative name
Primer information details of the primers used for amplification of the target sequences  Click to view information
Primer information details of the primers used for amplification of the target sequences  Click to hide information
Sequence Primer 1
Additional information primer 1
Sequence Primer 2
Additional information primer 2
Sequence Primer 3
Additional information primer 3
Sequence Primer 4
Additional information primer 4