Development of transgenic Elysia chlorotica as an Effective Living Bioindicator for Detecting Environmental ToxicantsJONATHAN LING

Introduction• Many threats are endangering aquatic environments

• The discharge of industrial and domestic wastewater has the most significant impacts on freshwater ecosystems, as well as agricultural production and human health

• Constant monitoring is essential to ensure timely response whenever damaging waste discharge events happen

Nature is full of thieves:Elysia chlorotica• Novel organism that

demonstrates high feasibility for horizontal gene transfer

• Observed phenomenon of kleptoplasty – Biological burglars

• Natural genetic engineering – Example of a natural transgenic process in the so-called higher life forms

Justifications of model• Ability to maintain the chloroplasts acquired from

Vaucheria litorea in a functional state demonstrates:

• Elysia chlorotica must possess photosynthesis-supporting genes within its own nuclear genome, most likely acquired through horizontal gene transfer

• Elysia chlorotica has incorporated the required algal DNA into its own DNA so the algal plastids (now Sea Slug plastids) can receive the proteins they need to function according to specification

• Vital algal gene, psbO, found in the sea slug's DNA. Found to be identical to the algal version

Methods• Introduction of GFP transgene regulated by the

upstream open reading frame (uORF) fragment of human DNA-damage-inducible transcript 3 (ddit3, previously named chop) cDNA

• uORF-based translational regulation plays significant, if not primary, roles in the production of CHOP protein, while the chop gene is one of the most commonly used biomarkers for endoplasmic reticulum (ER) stress

• Gene can be introduced into organisms and maintained in their genome through breeding, injection with a viral vector, or cell transformation

CHOP gene• The CHOP gene is induced by cellular stress and is

involved in mediating apoptosis

• Implicated in adipogenesis and erythropoiesis, is activated by endoplasmic reticulum stress, and promotes apoptosis

• Conserved region in the promoter of the CHOP gene responsible for its inducibility by endoplasmic reticulum (ER) stress

• The unfolded protein response (UPR) is a cellular stress response related to the endoplasmic reticulum. It is a stress response that has been found to be conserved between all mammalian species, as well as yeast and worm organisms

GFP Transgene• GFP gene is frequently used as a reporter of expression

• GFP gene has been introduced and expressed in many Bacteria, Yeast and other Fungi, fish, plants, flies, and mammalian cells

• Different stresses could cause different GFP expression patterns in a dose-dependent manner

• Holds considerable promise as a novel fluorescent biomonitoring method

Methods• Embryos will be used for preliminary in vitro research

• Embryos derived from the transgenic Elysia chloroticaonly to display fluorescent signals upon encountering stresses, with no detectable leakage under normal conditions

• Embryos can give a faithful account of cellular stresses

• Could potentially be used to detect various environmental contaminants, including heavy metals and endocrine-disrupting chemicals (EDCs)

Why transgenic?When…• Normal conditions, such as growth, survival rates and

egg hatchability, can be used as monitoring parameters.

• Quantifying the activity of enzymatic defenses in is a common approach to assess water quality

• Interpretation of the data obtained from these methods is limited by the fact that multiple physiological, genetic, and metabolic factors may simultaneously affect these multifunctional enzymes

Why transgenic?

Why not native gene promoters?

• the cyp1a1 promoter, which is induced by polycyclic aromatic hydrocarbons

• the heat-shock promoter, which is induced by heat and other stressors

Why transgenic?• These promoters only respond to specific forms of

stress, their advantages over traditional chemical analysis are not particularly significant

• A given stress with little harm to the animal may still induce the expression of a reporter gene controlled by the heat-shock promoter

• The reporter activity would have little relationship to the actual physiological stresses

• Hence, for an animal model to be a practical biomonitor, it must (1) respond to a wide range of pollutants with accuracy and sensitivity and (2) dynamically trace physiological stresses

Limitations• We cannot discriminate natural variability from changes

due to human impacts, thus limiting the applicability of bioindicators in heterogeneous environments

• Ethical issues

• Issue of releasing a transgene into the wild if the slugs are to be used as living bioindicators