Mackenzie L Klemek’s scientific contributions

Forward genetic screening is a powerful approach to assign functions to genes and can be used to elucidate the many genes whose functions remain unknown. Chemical mutagenesis is an unbiased and efficient method for generating point mutations in the founding generation of animals in a forward genetic screening experiment. Missense and nonsense mutations induced by chemical mutagenesis can lead to the generation of partial function, gain-of-function, or null alleles that underlie compelling phenotypes, but positional cloning of the underlying causative single base pair changes can be laborious and time-consuming, especially in large polymorphic genomes. Current methods use a bioinformatic mapping-by-sequencing approach which often identifies large genomic regions which contain an intractable number of candidate genes for testing. Here, we describe WheresWalker, a modern mapping-by-sequencing algorithm that identifies a mutation-containing interval and then supports positional cloning to refine the interval which drastically reduces the number of potential candidates allowing for extremely rapid mutation identification. We validated this method using mutants from a forward genetic mutagenesis screen in zebrafish for modifiers of ApoB-lipoprotein metabolism. WheresWalker correctly maps and identifies novel zebrafish mutations in mttp, apobb.1, and mia2 genes, as well as a previously published mutation in maize. Further, we use WheresWalker to identify a previously unappreciated ApoB-lipoprotein metabolism-modifying locus, slc3a2a.

Lipoprotein kinetics are a crucial factor in understanding lipoprotein metabolism since a prolonged time in circulation can contribute to the atherogenic character of apolipoprotein-B (ApoB)-containing lipoproteins (B-lps). Here, we report a method to directly measure lipoprotein kinetics in live developing animals. We developed a zebrafish geneticly encoded reporter, LipoTimer, in which endogenous ApoBb.1 is fused to the photoconvertible fluorophore Dendra2 which shift its emission profile from green to red upon UV exposure. By quantifying the red population of ApoB-Dendra2 over time, we found that B-lp turnover in wild-type larvae becomes faster as development proceeds. Mutants with impaired B-lp uptake or lipolysis present with increased B-lp levels and half-life. In contrast, mutants with impaired B-lp triglyceride loading display slightly fewer and smaller-B-lps, which have a significantly shorter B-lp half-life. Further, we showed that chronic high-cholesterol feeding is associated with a longer B-lp half-life in wild-type juveniles but does not lead to changes in B-lp half-life in lipolysis deficient apoC2 mutants. These data support the hypothesis that B-lp lipolysis is suppressed by the flood of intestinal-derived B-lps that follow a high-fat meal.