Date of Award

January 2021

Document Type

Open Access Thesis

Degree Name

Medical Doctor (MD)

Department

Medicine

First Advisor

Marc Hammarlund

Abstract

REGULATED APOPTOSIS IN CAENORHABDITIS ELEGANS DEVELOPMENT.Nathan Lifton, Alison Kochersberger, and Marc Hammarlund, Department of Genetics, Yale University School of Medicine, New Haven, CT. We have used chromosome engineering in C. elegans to develop a model animal in which cells normally undergoing developmental programmed cell death (DPCD) survive and are visibly marked. This system uses three elements: The first is a mutation in the apoptosis activator, ced-3, that drives developmental programmed cell death, such that cells normally killed by DPCD survive. The second is a cassette in which the gene encoding the bacterial enzyme Cre recombinase is expressed under transcriptional control of the regulatory elements of the egl-1 gene (Cre protein induces homologous recombination between specific DNA sequence elements called LoxP elements). The third element is a green fluorescent protein (GFP) reporter gene that is connected to a constitutively active promoter; however, GFP cannot be produced from this construct without the expression of Cre because two exons of GFP are inverted within the gene and flanked by inverted LoxP sites. When Cre recombinase is expressed, it will induce flipping of exons 2 and 3 of GFP, resulting in an intact GFP that is constitutively expressed, providing a permanent mark for cells in which expression at the egl-1 locus was activated even transiently. We succeeded in producing worms with the desired constructs by injecting gonads with linear extrachromosomal arrays that were integrated at random sites in the genome by induction of double strand DNA breaks with trimethylpsoralen and ultraviolet light. We developed multiple strains with independent insertions of the egl-1/Cre (4 independent insertions) and Cre-dependent GFP (3 independent insertions) cassettes, demonstrated that each had integrated into a single C. elegans chromosome, and performed crosses to produce worms that were homozygous at all three loci (ced-3, egl-1/Cre and Cre-dependent GFP). Preliminary analysis of the resulting worms from two of these strains provides evidence of Cre-dependent GFP expression, with reproducible expression of GFP in cells of the posterior bulb of the pharynx, which includes the head ganglia.

Comments

This is an Open Access Thesis.

Download
Open Access

This Article is Open Access

Share

COinS