This web page was produced as an assignment for Genetics 677, an undergraduate course at UW-Madison
LDLR RNAi
Phenotypic analysis of LDLR using RNAi databases was performed on similar genes in Caenorhabditis elegans and Drosophila melangaster. Using WormBase and BLAST the hypothetical protein T13C2.6 in C. elegans was found to be a fairly similar to human LDLR. Although a BLAST of the mRNA sequences gave a high E-value of 2.2, the amino acid sequences were much more similar (E-value=9e-117). The subsequent analysis will be performed using isoform T13C2.6a.1. For D. melanogaster, FlyBase and BLAST was used to find conserved genes within the fly. LpR2 isoform A was shown to be very similar to human LDLR gene. A BLAST of the mRNA sequences gave an E-value of 7e-08 and an E-value of 4e-165 was determined with amino acid sequences.
Caenorhabditis elegans Phenotype
The only observable RNAi phenotype for T13C2.6 on WormBase can result in embryonic lethality. The historical name of the RNAi is cenix:206-b9. Phenobank was then used to visualize this phenotype. One issue I had with the RNAi information on both WormBase and Phenobank was the inconsistencies of phenotype depending on the experiment. Although both websites state cenix:206-b9 treatment results in embryonic lethality some of the documented experiments have no effect on the phenotype. Also, depending on when the experiment is administered different phenotypes result. These phenotypes ranged from normal development to 50-70% embryonic lethality. I was especially surprised to find these inconsistencies of phenotype because the researchers used injection to incorporate the RNAi into the organisms. Injection is the most potent RNAi technique for gene knockdown experiments (1).
| small_206-b9-1-d1-1.mov |
The above movie is from Phenobank and is shows the cenix:206-b9 phenotype. The embryo seems to develop normally throughout the entirety of the movie. I think this is because the phenotype is not lethal in the early stages of C. elegans development. Also, the embryonic lethality phenotype is fairly inconsistent, with multiple mutants not giving the phenotype. This is reflected in the movie.
Drosophila melanogaster Phenotype
Searching for phenotypes in D. melanogaster was more difficult compared to C. elegans. FlyBase had a lot of information on LpR2, but there were no observable phenotypes for this gene. The only listed phenotype was for the LpR2 allele EY07715b. However, this allele did not really affect the phenotype; the offspring are viable and fertile. I attempted to find RNAi phenotypes in D. melanogaster using FLYRNAi.org. There was only one hit for LpR2, the RNAi DRSC13726. It was shown to induce an insulin response, but again this phenotype was difficult to actually find information about. I could only find the other RNAi's that caused an insulin response, but I could not find information on how this response affected the phenotype.
Danio rerio Phenotype
Since the above phenotypes are not very helpful, I searched the ZFIN database (zebrafish) for potential Danio rerio mutant phenotypes. Unfortunately, I was unable to find any relevant mutants or phenotypes in the Danio rerio ldlr gene.
Analysis
Although I was able to find similar genes to LDLR in C. elegans and D. melanogaster, I don't know how helpful these model organisms would be for studying FH. The RNAi phenotypes in both organisms did not show any similarities to mutations within human LDLR. This discovery brings up a good point: just because a gene encodes a similar amino acid sequence across many species does not mean it functions the same in those species. Cholesterol uptake is a complex process that should probably be studied in higher organisms, such as mice, in order to truly understand the mechanisms.
References
1. Sugimota, A. (2004). High-throughput RNAi in Caenorhabditis elegans: genome-wide screens and functional genomics. Differentiation, 72, 81-91. PMID: 15066188
