Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1998-08-13
2004-03-09
Crouch, Deborah (Department: 1632)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S320100, C435S325000, C435S455000
Reexamination Certificate
active
06703209
ABSTRACT:
This invention relates to totipotent porcine cells and to the recovery, proliferation and use thereof. The invention further relates to the use of a porcine stem cell factor (pSCF), which may also be termed porcine “steel” factor, which is preferably membrane-bound pSCF on transfected murine STO feeder cells, for establishing pluripotent or totipotent porcine cell lines, especially for the culture of porcine primordial germ cells (PGCs).
PGCs are precursor cells of the germ line in the developing embryo. In swine (porcine) embryos PGCs migrate from the base of the allantois through the hindgut epithelium and dorsal mesentery to colonize the gonadal anlage. During the migration to the genital ridge the PGCs increase in number in pigs to approximately 1×10
6
by day 50. Porcine PGCs can be distinguished from somatic cells by their morphology: large nuclei, high nuclei-cytoplasm-ratio, occasional blebbing and by clearly visible pseudopodia.
Totipotent cell lines have been obtained from primordial germ cells (PGCs) in mice. Such pluripotent or totipotent cell lines are important in the field of transgenic animals. Several methods for producing transgenic animals, including a microinjection method, exist and are well-known in the art. However, a drawback to the microinjection method is that providing transgenic animals into whom genes can be embedded at will in their chromosomes is difficult.
Fortunately, producing such transgenic animals is made potentially possible by using totipotent or pluripotent cells such as embryonic stem (ES) cells to establish a transgenic animal line. For example, ES cells can be injected into a mouse embryo near the embryonic location which will give rise to germ cells in the resulting adult mouse, and result in functional germ cells originating from the injected ES cells. Therefore, such adult mouse can produce offspring that have the traits of the injected ES cells. If the ES cells have been altered genetically prior to their injection into the embryo such offspring of the resulting adult mice can form a chimeric line of mice with the genetically altered trait.
Accordingly, an ES cell line maintained in culture in a mouse can be utilized for a variety of gene introduction methods (for example, the retrovirus vector method, the electroporation method and the calcium phosphate method) and then injected into embryos as part of a process to produce chimeras with desired altered genetic traits. With such a procedure it is possible to obtain an individual which has been altered at a specific gene locus by substitution of a desired gene, whether active or inactive, for the original gene by homologous recombination following insertion of the gene into the ES cell.
Through the totipotent-cell-embryo-injection-method precise alteration of a single gene trait and the study of a specific gene function in a resulting chimeric animal line is possible.
Also, in such chimeric lines it would be possible through such precise genetic manipulation to cause tissue protein changes and remove certain native antigens so that an organ or tissue from the chimeric animal could be transferred to another species without rejection by the other species.
In mice, primordial germ cells isolated during their migratory phase and cultured on feeders layers (e.g., fetal fibroblast cells (“STO cells”), Kawase et al.
Experimental Medicine
, Vol. 10, No. 13, 1575 to 1580 (1992)) with leukaemia inhibitory factor (LIF) and mouse stem cell factor (MSCF) provide mice PGCs that result in cell lines for long-term culture. In addition, mice PGCs can also be cultured in similar media to which basic fibroblast growth factor (bFGF) has been added, thereby converting PGCs to cells that resemble undifferentiated embryonic stem cells (ESs) (Matsui et al., Cell 1992 70:5:841-847; Resnick et al., Nature 1992 359 (6395):550-551). Such (embryonic germ cell derived lines (“EGs”) derived from PGCs can be utilized to contribute to the germ-line of chimeric mice. Early culture mice PGCs, cultured in the presence of LIF and MSCF, can be observed as burst colonies of cells with a flattened and polarized morphology that are characteristic of motile cells. By contrast, mice ESs or EGs derived from PGCs (e.g., long-term cultures) tend to form discrete colonies of tightly packed cells and resemble in morphology the germ cells observed in vivo when PGCs migrate to the germ layers in the embryo, differentiate into germ cells and establish colonies in the germ layer.
Quiescent descendants of ESs or EGs (long term cultures or germ cells from differentiated PGCs), which are observed to form tightly packed colonies rather than burst colonies, often require less growth factors or simple nutrients to continue proliferating. These totipotent germ cell lines, thus established, can be maintained in culture (by recycling to fresh media) and used to inject embryos, or alternatively can be frozen, thawed and then used.
Authors of work with mice PGCs, ESs or EGs have hypothesized that results with mice embryos could lead to similar results with other species. They hypothesized that it might be possible to use PGCs from other species along with such mice feeder cells which produce MSCF and LIF (such as transfected murine STO cells) for the establishment of totipotent cell-lines in domestic animals.
The classical ways of establishing pluripotent embryonic stem cells involve culturing of a preimplanted embryo or an enucleated egg which has been implanted with an isolated inner cell mass (ICM). Although this is routinely performed in mice, unfortunately, modified protocols in attempts to establish similar lines in domestic animals such as pigs have not been very successful. Such is evidenced by the inability of such lines to colonize somatic tissues or to provide a germ line of chimeric animals. This may be due to the fact that, unlike the mouse, ungulates have slowly proliferating early embryos with very low cell numbers. Therefore, until now, obtaining cell lines from domestic animals such as pigs has not been done successfully. Whether such procedures would even work with PGCs from domestic animals has remained an open and debatable question in the scientific community.
Accordingly, there is a need for establishment of totipotent/pluripotent cell lines from domestic animals, for example swine. Relative to other domestic animals, swine (or pigs) are easy to feed and maintain. Also, they have no reproductive season, can produce a fairly large number of fetuses, and provide offspring after only a relatively short gestation period.
Establishment of porcine pluripotent or totipotent PGC lines as an alternative to embryonic stem cells (ESCs) would be useful for studies of cell differentiation and gene regulation during embryonic development. Such lines would be important for the generation of transgenic pigs especially for their use in gene farming or xenotransplantation.
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Baetscher Manfred
Brem Gottfried
Biotransplant, Inc.
Crouch Deborah
Lillie Raymond J.
Olstein Elliot M.
Woitach Joseph
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