Generation of Transgenic Hydra by Embryo Microinjection

Summary

Stably transgenic Hydra are made by microinjection of plasmid DNA into embryos followed by random genomic integration and asexual propagation to establish a uniform line. Transgenic Hydra are used to track cell movements, overexpress genes, study promoter function, or knock down gene expression using RNAi.

Abstract

As a member of the phylum Cnidaria, the sister group to all bilaterians, Hydra can shed light on fundamental biological processes shared among multicellular animals. Hydra is used as a model for the study of regeneration, pattern formation, and stem cells. However, research efforts have been hampered by lack of a reliable method for gene perturbations to study molecular function. The development of transgenic methods has revitalized the study of Hydra biology¹. Transgenic Hydra allow for the tracking of live cells, sorting to yield pure cell populations for biochemical analysis, manipulation of gene function by knockdown and over-expression, and analysis of promoter function. Plasmid DNA injected into early stage embryos randomly integrates into the genome early in development. This results in hatchlings that express transgenes in patches of tissue in one or more of the three lineages (ectodermal epithelial, endodermal epithelial, or interstitial). The success rate of obtaining a hatchling with transgenic tissue is between 10% and 20%. Asexual propagation of the transgenic hatchling is used to establish a uniformly transgenic line in a particular lineage. Generating transgenic Hydra is surprisingly simple and robust, and here we describe a protocol that can be easily implemented at low cost.

Introduction

Hydra has been used to study regeneration, pattern formation, and stem cells for approximately 250 years². Hydra has a simple body plan consisting of three cell lineages: ectodermal epithelial, endodermal epithelial, and interstitial. The tubular body is formed by the ectodermal and endodermal epithelial lineages, each of which is a single cell layer. All of the epithelial cells in the body column are mitotic. When epithelial cells are displaced into the extremities³, the head (mouth and tentacles) at the oral end or the foot (basal disc) at the aboral end, they arrest in the G2 phase of the cell cycle and change cell fate^4....

Protocol

1. Preparation of Plasmid DNA, Needles, and Injection Dishes

1. Prepare the plasmid DNA using a High Speed Maxi or Midi kit and concentrate the DNA to 2.5 mg/ml using ethanol precipitation. The DNA pellet should be dissolved in nuclease-free deionized water. Store the DNA in 10-20 µl aliquots at -20°C. (See Supplementary Table 1 for a list of available vectors)
2. Make an injection dish using agarose to construct a trough for holding embryos in a 100 x 15 mm Petri dish.
   1. Place a 75 x 50 glass microscope slide into a 100 x 15 mm Petri dish at an angle. Pour approximately 50 ml of 2% agarose melted in Hydra ....

Results

Establishing transgenic Hydra lines

Feed transgenic hatchlings every 2-3 days with Artemia nauplii. Hatchlings sometimes do not eat for a day or two after hatching. Some new hatchlings will never eat, and thus will not survive. If the hatchling is transgenic in either the ectodermal (Figure 1A) or endodermal epithelial tissue, allow the animal to bud and collect the buds that have the most transgenic tissue (Figure 1B, C). Continue to d.......

Discussion

Hydra routinely reproduces asexually, but requires environmental stimuli to begin producing gametes. These stimuli are not well-defined for most Hydra species and may differ from strain to strain. A significant hurdle to the production of transgenic Hydra is obtaining embryos on a regular basis because it can be difficult in a laboratory setting to induce Hydra to become sexual. The AEP strain²⁵, however, produces gametes readily in the laboratory and this is the only strain.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
AEP Hydra Strain			Email: Celina Juliano at celina.juliano@yale.edu or Hiroshi Shimizu bubuchin2006@yahoo.co.jp
High Speed Maxi Kit	Qiagen	12662
100 x 15 mm Petri Dishes	BD Falcon	351029
75 x 50 mm Glass Microscope Slide	Sigma	CLS294775X50
Microinjection Fish Mold	IBI Scientific	FM-600
Borosilicate Glass with Filament	Sutter Instrument	BF100-50-10	O.D.: 1.0 mm, I.D.: 0.50 mm, 10 cm length
Flaming/Brown Micropipette Puller	Sutter Instrument	P-97
Phenol Red	Sigma	P3532
Jewelers Forceps, Durmont #5	Sigma	F6521
Scalpel Blade #15	Fisher	50-822-421
Mineral oil	Sigma	M8410-500ML
Microinjector	Narishige	IM-9B
Magnetic Stand	Narishige	GJ-1
Iron Plate	Narishige	IP
Joystick Micromanipulator	Narishige	MN-151