Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving virus or bacteriophage
Reexamination Certificate
2001-01-13
2002-07-23
Wang, Andrew (Department: 1635)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving virus or bacteriophage
C435S006120, C536S023100, C425S288000
Reexamination Certificate
active
06423488
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates generally to a screening assay and, more specifically, to a high-throughput screening assay useful for detecting the presence of a foreign DNA sequence in a sample. The present invention further includes a high throughput extraction method for extracting DNA from nucleated cells, particularly red blood cells.
BACKGROUND OF THE INVENTION
The present invention provides a high throughput screening assay useful for detecting the presence of an exogenous DNA sequence in a sample. The method of the present invention further includes a high throughput DNA extraction method useful for extracting DNA from avian blood for subsequent use in a screening assay as, for example, an assay to detect the insertion of foreign DNA in the genome of a recipient.
The publications cited herein to clarify the background of the invention and in particular, materials cited to provide additional details regarding the practice of the invention, are incorporated herein by reference, and for convenience are cited in the following text.
Transgenesis is the ability to introduce foreign or exogenous DNA into the genome of a recipient, as for example, into a sheep, a cow or even a chicken. The ability to alter the genome of an animal immediately suggests a number of commercial applications, including the production of an animal able to express an exogenous protein in a form that is harvested easily.
The main obstacle to avian transgenesis is the low efficiency of introduction of foreign DNA into the chicken genome. The insertion of foreign DNA into the chicken genome using procedures that have worked for other animals is a difficult task and attempts at such have been mostly unsuccessful, partly due to the unique physiology of the chicken (Love et al., Transgenic birds by DNA microinjection,
Biotechnology
12: 60-63, 1994; Naito et al., Introduction of exogenous DNA into somatic and germ cells of chickens by microinjection into the germinal disc of fertilized ova,
Mol Reprod Dev
37: 167-171, 1994).
Through the use of retroviruses, a number of research groups have successfully introduced foreign DNA into the chicken genome at acceptable but low efficiencies (Bosselman et al., Germline transmission of exogenous genes in the chicken,
Science
243: 533-5, 1989; Petropoulos, et al., Appropriate in vivo expression of a muscle-specific promoter by using avian retroviral vectors for gene transfer [corrected] [published erratum appears in
J Virol
66: 5175, 1992]
J Virol
66: 3391-7, 1992; Thoraval. et al., Germline transmission of exogenous genes in chickens using helper-free ecotropic avian leukosis virus-based vectors,
Transgenic Res
4: 369-377, 1995). The retroviral vectors used have been engineered such that they will not result in the replication and spread of any new retroviruses. This allows production of transgenic chickens that are free of any retrovirus. However, because the retroviral vectors cannot propagate in the chicken, the transgene is not transmitted from cell to cell. Retroviral vectors are typically injected into the embryo of a freshly laid egg through a small window in the egg shell. Approximately 1% of the embryonic cells are transduced, such that one copy of the transgene is inserted into the cell's DNA. After sexual maturity and meiosis, 0.5% of sperm or oocytes carry the transgene. In order to obtain one transgenic bird, at least 200 chicks have to be screened. It is often desirable to obtain several transgenic chicks because different chromosomal insertions can lead to different levels of transgene expression. Thus, it is necessary to breed and screen hundreds to thousands of chicks, necessitating a method for high throughput genetic screening for detecting the desired genetic sequence.
Random chromosomal insertion of transgenes via non-retroviral methods has become the mainstay of transgenics in some domesticated animals including pigs, sheep, goats and cows. The primary method to introduce the transgene is the injection of linearized DNA containing the desired transgene into the pronucleus of a zygote. Up to 20% of G
o
offspring contain the transgene. The relative high efficiency of transgenesis offsets the high technical costs incurred during the procedure. Transgenes have been inserted into goats, for instance, that direct the expression of pharmaceuticals in mammary glands for subsequent secretion into milk (Ebert, et al., Transgenic production of a variant of human tissue-type plasminogen activator in goat milk: generation of transgenic goats and analysis of expression,
Biotechnology
9: 835-8, 1991).
In chickens, injection of the zygote germinal disk has been accomplished but with limited success, in part due to additional complications associated with unique aspects of chicken physiology and embryogenesis (Love et al., 1994; Naito et al., 1994). One lab has successfully produced several transgenic chickens, which have incorporated the injected DNA into their chromosomes and passed the transgene on to offspring. Another lab attempted to reproduce the technique but failed. Zygote injections in chickens are difficult because the nucleus is very small and is about 50 microns below the yolk membrane. Thus, the DNA must be injected into the cytoplasm. As in mice, cytoplasmic injection of DNA results in inefficient incorporation of the transgene into the chromosomes. Chickens must be sacrificed in order to remove the zygote and each chicken yields only one zygote.
An important technical breakthrough was pioneered by Gibbins, Etches, and their colleagues at the University of Guelph by using blastodermal cells (BDCs) collected from embryonic stage X embryos at oviposition, e.g., the time when the egg is laid (Brazolot et al., Efficient transfection of chicken cells by lipofection, and introduction of transfected blastodermal cells into the embryo,
Mol Reprod Dev
30: 304-12. 1991; Fraser, et al., Efficient incorporation of transfected blastodermal cells into chimeric chicken embryos,
Int J Dev Biol
37: 381-5, 1993). Coupled with recent progress in the culturing of BDCs, which can still reconstitute the germline, the method theoretically enables random transgene addition via nonhomologous recombination as well as targeted gene engineering via homologous recombination.
At stage X, the embryonic blastoderm consists of 40,000 to 60,000 cells organized as a sheet (area pellucida) surrounded by the area opaca; it harbors presumptive primordial germ cells (PGCs) that have not yet differentiated into migrating PGCs. Dispersed BDCs can be transfected with an appropriate transgene and introduced into the subgerminal cavity of y-irradiated, recipient stage X embryos. Irradiation may selectively destroy presumptive PGCs and retard recipient embryo growth allowing injected cells additional time to populate the recipient blastoderm. Using genetic markers for feather color (black for Barred Rock and white for White Leghorn), Etches, Gibbins and their colleagues were able to show that, of injected embryos surviving to hatch, 50% or greater of these were somatic chimeras of which nearly half were also germline mosaics (Petitte, et al., Production of somatic and germine chimeras in the chicken by transfer of early blastodermal cells,
Development
108: 185-9, 1990).
Gibbins and her colleagues have determined that random gene addition occurs in in vitro cultured BDCs in 1 out of every 300 transfected cells (Gibbins and Leu, personal communication). They did not determine whether BDCs with random gene additions can be re-introduced into stage X embryos to produce germline G
o
chimeras. Therefore, the actual efficiency of transgenesis has not yet been determined.
Gene targeting, the ability to specifically modify a specific gene, is a much sought-after technology in a variety of species, including chickens, because such modifications will result in very predictable transgene expression and function. Gene targeting has been successfully accomplished in mice because mouse embryonic stem (ES) cells can be cultured in vitro fo
AviGenics, Inc
Jarecki-Black Judy
Lacourciere Karen A.
Wang Andrew
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