Multicellular living organisms and unmodified parts thereof and – Method of making a transgenic nonhuman animal
Reexamination Certificate
1999-03-22
2002-08-13
Priebe, Scott D. (Department: 1632)
Multicellular living organisms and unmodified parts thereof and
Method of making a transgenic nonhuman animal
C536S023100, C536S023500, C435S455000
Reexamination Certificate
active
06433247
ABSTRACT:
The invention is concerned with a method of introducing nucleic acid into nematode worms, in particular
Caenorhabditis elegans.
Transgenic
Caenorhabditis elegans
(
C. elegans
) are currently made by injecting nucleic acid (usually DNA) into the hermaphrodite gonad (i.e. into a syncitium)or into individual oocyte nuclei. Typically one injects a mixture of the DNA one wants to introduce (hereinafter referred to as ‘test DNA’) and a plasmid carrying a selectable marker that allows one to distinguish transgenic progeny from non-transgenic progeny. The selectable marker can be a visible phenotypic marker which leads to a change in shape or movement of the transgenic worms (e.g. rol-6), a marker rescuing a conditionally lethal gene introduced into the genetic background of the injected worms or a plasmid containing nucleic acid encoding green fluorescent protein (GFP) from the jellyfish
Aequorea victoria.
The offspring of injected worms (F1 generation) are then screened for animals expressing the selectable marker.
The F1 offspring of an injected hermaphrodite typically contain on average 1 to 10 individuals that express the selectable marker. These individuals are then placed in culture but on average only 10% will go on to transmit the selectable marker to their offspring. One generally assumes that when the marker DNA is accepted into the worm genome and transmitted to the offspring, the test DNA which one wants to introduce is co-transformed.
This current method of transformation has practical limitations in that introducing DNA into worms entails one by one manipulation and injection of syncitia/oocytes under a microscope. This work is time consuming and requires a considerable amount of expertise. It is typically possible for a person skilled in the injection technique to inject ~50 hermaphrodites in a single day.
Transgenic worms transmitting the transformed DNA in a heritable manner have incorporated an extra minichromosome, consisting of a mixture of marker and test DNA linked together in an unpredictable structure. This minichromosome is mitotically and meiotically unstable and is lost at a rate of 1% to 99% per cell division.
It is an object of the present invention to provide a more efficient transformation system for
C. elegans.
Ideally one would wish to achieve integration of the exogenous test DNA by homologous recombination with the
C. elegans
chromosome. In order to achieve this aim it will be necessary to develop a technique by which DNA can be simultaneously introduced into a large number i.e. thousands of individual worms.
A recently developed method for introducing DNA into cells involves shooting cells with microprojectiles, typically gold or tungsten particles of around 2 &mgr;m diameter, which have been coated with the DNA to be introduced. This technique, generally known to persons skilled in the art as ballistic transformation, has been used to successfully deliver DNA into plant cells (Klein et al. Nature, 327: 70-73 (1987); Christou et al. Plant Physiol., 87: 671-674 (1988); Takeuchi et al. Plant Molecular Biology, 18: 835-839 (1992)), cultured mammalian cells (Zelenin et al. FEBS Letters, 244: 65-67 (1989)), fertilized fish eggs (Zelenin et al. FEBS Letters, 287: 118-120 (1991)) and intact mouse tissues and organs (Zelenin et al. FEBS Letters, 280: 94-94 (1991); Williams et al. Proc. Natl. Acad. Sci. USA, 88: 2726-2730 (1991)).
Despite the success of the technique with plant cells and cultured mammalian cells problems have been anticipated by those skilled in the art in applying ballistic techniques to nematode worms. However, the present inventors have successfully applied a similar ballistic transformation technique to the introduction of nucleic acid into
C. elegans.
Using this technique it is possible to introduce nucleic acid simultaneously into a large number of individual worms.
Accordingly, in a first aspect the invention provides a method of introducing nucleic acid (DNA and/or RNA) into a nematode worm comprising bombarding the worm with a plurality of microprojectiles.
In one embodiment of the invention the microprojectiles are coated with the nucleic acid which it is desired to introduce into the nematode worm.
Bombardment of the nematode worm with high velocity microprojectiles is achieved using a particle bombardment gun based on flowing helium of a type known in the art, see for example Johnston, Nature, 346: pp776; Klein et al. Biotechnology, 10: pp286-291 and Takeuchi et al. Plant Mol. Biol., 18: pp835-839. The gun uses a flowing stream of helium gas to accelerate DNA coated particles towards a target sample to be transformed.
A detailed protocol for ballistic transformation of
C. elegans
using nucleic acid coated microprojectiles is described in the examples given herein. Briefly, a small pellet of worms is dispensed onto a small nematode agar plate. The plate is then placed inside the ‘gun’ and a suspension of microprojectiles (e.g. gold particles) coated with nucleic acid is shot at the worms. After a short recovery period the plate is cut into a number of segments which are placed on large agar plates to grow worms for selection of transgenic animals. The transformation procedure takes only a few minutes and is technically very simple so that a large number of experiments can be undertaken in very little time.
In an alternative embodiment of the method of the invention ballistic transformation can also be accomplished by first applying a solution containing the nucleic acid directly onto the nematodes and then shooting the nematodes with ‘bare’ microprojectiles which have not been coated with nucleic acid. With this technique it is not necessary to coat the microprojectiles with nucleic acid. Using the conventional bombardment technique (i.e. using coated microprojectiles) transformed offspring are produced as a result of a coated particle being fired into a gonad cell of the worm. Using the alternative approach, in which the worms are first coated with a dense solution of nucleic acid and then bombarded with ‘bare’ microprojectiles, a particle may drag the DNA along its passage through the worm and hence the particle does not necessarily need to stop within a gonad cell. If the particle merely passes through a gonad cell on its passage through the worm it may leave behind a sufficient amount of the nucleic acid it is dragging along to result in transformation of the gonad cell.
In order to facilitate selection of transformants into which DNA has been successfully introduced by the method of the invention it is preferred to use a dual selection protocol using a dominant phenotypic marker such as, for example, rol-6 or an autonomous fluorescent protein (AFP) in combination with a marker rescuing a conditionally lethal gene introduced into the genetic background of the injected worms. As used herein the term “autonomous fluorescent protein” encompasses both green fluorescent protein (GFP) and blue fluorescent protein (BFP) and any other autonomous fluorescent protein of this type. The examples given below relate to the transformation of
C. elegans
with a genetic background carrying a temperature sensitive mutation in the pha-1 gene wherein DNA encoding the wild-type pha-1 gene is introduced as a co-selectable marker. However, other conditional lethal mutations could have been used with equivalent effect and it is to be understood that the present invention is not to be limited by the nature of the selectable markers employed to facilitate the identification of transformed worms.
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Wittmann et al., Development, vol. 124, pp. 41
Chen Shin-Lin
Devgen N.V.
Priebe Scott D.
Wolf Greenfield & Sacks PC
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