[go: up one dir, main page]

WO1996000285A1 - Nouveaux animaux et nouvelles lignees cellulaires - Google Patents

Nouveaux animaux et nouvelles lignees cellulaires Download PDF

Info

Publication number
WO1996000285A1
WO1996000285A1 PCT/US1995/007255 US9507255W WO9600285A1 WO 1996000285 A1 WO1996000285 A1 WO 1996000285A1 US 9507255 W US9507255 W US 9507255W WO 9600285 A1 WO9600285 A1 WO 9600285A1
Authority
WO
WIPO (PCT)
Prior art keywords
gene
interest
human animal
cells
cell line
Prior art date
Application number
PCT/US1995/007255
Other languages
English (en)
Inventor
Robert H. Whitehead
Joan L. Joseph
Original Assignee
Ludwig Institute For Cancer Research
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Ludwig Institute For Cancer Research filed Critical Ludwig Institute For Cancer Research
Priority to AU28195/95A priority Critical patent/AU2819595A/en
Publication of WO1996000285A1 publication Critical patent/WO1996000285A1/fr

Links

Classifications

    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0271—Chimeric vertebrates, e.g. comprising exogenous cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/0331—Animal model for proliferative diseases
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells
    • C12N2510/04—Immortalised cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2517/00—Cells related to new breeds of animals
    • C12N2517/02—Cells from transgenic animals

Definitions

  • NOVEL ANIMALS AND CELL LINES This invention relates to animals bearing an immortalizing gene together with a gene of interest, and cell lines capable of long term growth in vitro.
  • the invention relates to conditionally immortalized cell lines derived from mice carrying the Multiple Intestinal Neoplasia gene, which predisposes to colon cancer, and a temperature sensitive mutant of the SV40 large T gene which allows growth at certain temperatures.
  • Conditionally immortalized cell lines have been established from small intestinal epithelivu ⁇ , colonic mucosal epithelium, liver epithelium and stomach epithelium. Fibroblast cell lines from small intestinal stroma, colon, muscle and skin have also been established. These animals and cell lines are useful in studies of aberrations of growth and differentiation, including carcinogenesis that may be induced, for example, by viruses, various genes or mutagens.
  • Familial Adenomatous Polyposis is a genetically-determined condition resulting from a mutation of the Adenomatous Polyposis Coli (APC) gene. Individuals with Familial
  • Adenomatous Polyposis have a greatly increased propensity to develop colon cancer, and mutations of the APC gene are also found in patients with non-familial (ie. sporadic) colon cancer. With the development of the Multiple Intestinal
  • Neoplasia (Min) mouse (1) a mouse model of Familial Adenomatous Polyposis in humans became available (2-4) .
  • the usefulness of this model was enhanced by the finding that the Min mutation is a nonsense mutation in codon 15 of the mouse APC gene (5) .
  • This gene is the mouse homologue of the human APC gene (3) which is mutated in most Familial Adenomatous Polyposis patients (6,7) .
  • the Min mouse has been shown to carry a dominant mutation of the murine homologue of the human APC gene, which expresses its phenotype in all mice bearing the mutant allele as multiple intestinal tumours, in both small intestine and colon, at an early age, usually by 120 days (1) .
  • Mutations of the APC gene comprise one of a series of mutations and deletions in different genes that occur during the development of both familial and sporadic colon cancer in humans (6,7,18).
  • the APC gene appears to become mutated early in the sequence from normal colonic epithelium to adenoma to malignancy that occurs in colon cancer (18) .
  • a second gene, the tumour suppressor gene Deleted in Colon Carcinoma (DCC), which is a gene mutated in both alleles of chromosome 18q, is present in approximately 70% of human colon carcinomas.
  • the protein encoded by this gene is similar in structure to members of the cellular adhesion molecule (CAM) group of proteins, which are known to be involved in processes of development and differentiation.
  • Another tumour suppressor gene, the p53 gene in chromosome 17 is also an important contributor to the oncogenic phenotype in colon cancer.
  • Murine analogues of these genes have been at least partially cloned and characterised.
  • the intestinal epithelial cell lines expressed known brush border-associated enzymes (11) .
  • the Immortomouse strain has now been used to establish Fl Min/Immortomouse hybrids which carry both the SV40 large T gene and the Min mutation. Described herein is the establishment of a conditionally immortalized epithelial cell line from the colonic mucosa of an Fl hybrid mouse which carries the Min allele. It was necessary to use a hybrid strain carrying an immortalizing gene, as it was not possible to culture the normal colonic epithelial cells of adult animals for more than a few days (8,9,19). Conditionally immortalized Min/+ cell lines have also been derived from the liver and small intestinal stroma of the same mouse, and other conditionally immortalized Min/+ cell lines have been established.
  • the Immortomouse is useful for developing cultures from tissues, such as the intestine, that have previously proved very difficult to culture in vitro.
  • the invention provides a generally-applicable method for obtaining cell lines of a desired mammalian tissue carrying one or more genes of interest, using the same principle, a cell line lacking a gene of interest can be obtained.
  • An animal either carrying or deficient in a gene of interest is crossed with an animal of the same species carrying an immortalizing gene in its germ line.
  • the Immortomouse can be crossed with any other transgenic mouse strain carrying a gene of interest, to obtain progeny expressing both the SV40 large T gene and the gene of interest.
  • the Immortomouse can be crossed with a mouse bearing the Ras oncogene.
  • the Immortomouse can be crossed with a mouse in which a gene of interest has been specifically disrupted, to obtain immortalized cells in which the gene of interest is not expressed.
  • the Immortomouse can be crossed with a mouse in which a gene for a growth factor such as transforming growth factor ⁇ , fibroblast growth factor, or epidermal growth factor is disrupted. Organs from these mice will yield tissue that can be cultured in vitro.
  • the animals and cell lines of the invention are especially useful in studies such as: a) detection of tissue specific markers; b) effects of growth factors on cells of different tissues; c) effects of oncogenes associated with cancers of different tissues; d) screening of putative initiators, promoters and inhibitors of malignant progression; e) screening of putative therapeutic agents; f) screening of putative growth factors; g) the effect of the gene(s) of interest or its absence on differentiation, senescence and cell death in tissues which undergo these processes; h) effect of possible modifiers on differentiation, senescence and cell death, in tissues bearing or lacking the gene of interest; and i) cell-cell interactions between cells of different lineages.
  • the invention provides an Fl hybrid non-human animal, of which one parent carries an immortalizing gene, and the other parent carries one or more genes of interest.
  • the immortalizing gene may be any gene which confers the capacity for long-term growth in cell culture. Suitable immortalizing genes include, but are not limited to, the SV40 large T gene, the adenovirus ElA gene, and the polyoma virus middle T gene. Combinations of immortalizing genes may be used. Other suitable genes will be known to those skilled in the art. The only requirement is that the immortalizing gene is present in the germ line of one of the parental strains, and consequently is also present in all somatic cells of that parental strain.
  • the animal is a mammal, preferably a rodent, especially a rabbit, rat or mouse.
  • the gene of interest may be any gene for which a transgenic animal of the appropriate species can be generated, and is preferably one which confers a predisposition towards development of abnormalities such as cancer.
  • the gene of interest is preferably a cancer- associated gene, an oncogene, a tumour suppressor gene, or a tumour inhibitor gene.
  • the gene may bear one or more mutations, and/or it may be a transgene, including a transgene of human origin.
  • the gene of interest is selected from the group consisting of Multiple
  • Intestinal Neoplasia Min and its analogues, Adenomatous Polyposis Coli (APC), Deleted in Colon Carcinoma (DCC), or from the group consisting of Ras, Myb, Myc, Raf, p53 and pl6.
  • the gene of interest is the Multiple Intestinal Neoplasia gene, or its homologue in other animals.
  • the animal carries both a cancer- associated gene and an oncogene.
  • the gene of interest may be any gene for which a disruption of expression can be generated and transmitted via the germ line in a so-called “knockout” animal.
  • Many "knockout" animals are now known, particularly knockout mice, including many mouse strains in which growth factors or cytokines are not expressed.
  • the gene is preferably one encoding a cytokine, growth factor, hormone or enzyme.
  • Preferred such cytokines and growth factors include, but are not limited to, interleukins, interferons, epidermal growth factor, fibroblast growth factor, and transforming growth factor, particularly transforming growth factor ⁇ .
  • an animal in which the expression of a gene of interest is selectively disrupted will be referred to as an animal lacking that gene of interest.
  • the invention provides a Fl hybrid animal, of which one parent carries a temperature-sensitive mutant of the SV40 large T gene, and the other parent carries or lacks a gene of interest.
  • the parental strain carrying the temperature-sensitive mutant of the SV40 large T gene is a rodent, for example the Immortomouse (H-2K b -tsA58 SV40 large T) .
  • the invention provides an immortalized or conditionally immortalized cell line derived from an animal according to the invention.
  • the cell line may be derived from any tissue of the animal which is capable of growth in cell culture, and epithelial cells and fibroblasts are especially suitable. - 7 -
  • the cell line is selected from the group consisting of epithelial cells such as colonic epithelial cells, liver epithelial cells, small intestinal epithelial cells, and fibroblasts such as small intestinal fibroblasts, muscle fibroblasts and skin fibroblasts.
  • epithelial cells such as colonic epithelial cells, liver epithelial cells, small intestinal epithelial cells, and fibroblasts such as small intestinal fibroblasts, muscle fibroblasts and skin fibroblasts.
  • the cell line may be transfected with further genes, for example, the Ras oncogene.
  • the cells of these cell lines carry both the SV40 large T gene and one or more genes of interest.
  • the gene of interest is selected from the group consisting of Multiple Intestinal Neoplasia (Min) , Adenomatous Polyposis Coli (APC) , Deleted in Colon Carcinoma (DCC), Ras, Myb, Myc, Raf, p53 and pi6.
  • the gene of interest is the Multiple Intestinal Neoplasia gene.
  • the genes of interest are the Multiple Intestinal Neoplasia gene and the Ras oncogene.
  • the invention provides a method of obtaining an immortalized or conditionally-immortalized cell line carrying or lacking one or more genes of interest, comprising the steps of crossing a non-human animal bearing an immortalizing gene with an animal of the same species bearing or lacking the gene of interest to obtain an Fl hybrid animal, and culturing cells from the Fl hybrid animal under conditions suitable for growth thereof.
  • this aspect of the invention provides a method of obtaining a conditionally-immortalized cell line carrying or lacking one or more genes of interest, comprising the steps of crossing a mouse carrying or lacking the gene(s) of interest with a mouse carrying a temperature sensitive mutant of the SV40 large T gene to obtain an Fl hybrid mouse, and culturing cells from the Fl hybrid mouse At the permissive temperature.
  • the invention provides a method of screening agents suspected of inducing or predisposing to development of cancer, such as mutagens, oncogenes, carcinogens, co-carcinogens, viruses and growth factors, or of agents suspected to be useful in treatment or prevention of cancer, such as therapeutic agents, immunological response modifiers, receptors, hormones, antibodies, cytokines, and growth factors, comprising the step of exposing an animal or cell line of the invention to the agent to be tested.
  • This aspect of the invention also includes within its scope the use of cell lines of the invention derived from different tissues of the same animal to identify the role of tissue-specific elements in the response to such agents.
  • tumours targeted are preferably solid tumours such as colon, breast, bladder or ovarian tumours. Tissues from a tissue bank or from a fresh surgical sample may be used.
  • Figure 1 shows a phase contrast micrograph of colonic epithelial cells cultured from crypts isolated from the colonic mucosa of a young adult Immortomouse/Min hybrid mouse (Magnification is xl25);
  • Figure 2 illustrates staining of colonic epithelial cells at passage 6 with a monoclonal antibody specific for keratin 18 (LE61) (Magnification is x600);
  • Figure 3 shows a phase contrast micrograph of liver epithelial cells cultured from the liver of the Immortomouse/Min liver (Magnification is xl25);
  • Figure 4 shows the results of an assay demonstrating the conditional immortalisation of the colonic epithelial cells.
  • the cells were cultured at either the permissive temperature (33°C) or the non-permissive temperature (39.5°C) for 6 days, both with and without mouse ⁇ -interferon (IFN; 10 units per ml) .
  • the cell numbers in each well were counted. The results shown are the means of triplicate cultures;
  • Figure 5 shows the PCR products of cell lines derived from an Immortomouse/Min hybrid mouse. Lanes marked 1 to 7: reactions using the SV40 large T primers;
  • Lanes 8 to 14 PCR products derived from the Min oligonucleotides
  • Lanes 1 and 8 negative controls with no DNA
  • LLaanneess 22 aanndd 99 : derived from DNA prepared from mice which carried neither SV40 large T nor the mutated Min allele;
  • Lanes 4 and 11 DNAs from the colonic epithelial cell line from the Immorto/Min mouse cell lines
  • Lanes 5 and 12 fibroblasts derived from the small intestine.
  • Figure 6 shows the results of analysis of YAMC-Ras and MCE-Ras colonic cell lines for protein expression and genotyping.
  • Figure 7 shows the analysis of growth kinetics of YAMC-Ras and IMCE-Ras cell lines under a) permissive and b) non-permissive culture conditions.
  • Figure 8a shows the morphology of cell lines: i) YAMC cells, ii) YAMC-Ras cells, iii) IMCE cells, and iv) IMCE-Ras cells;
  • Figure 8b shows the growth of cell lines in soft agar: i) YAMC cells, ii) YAMC-Ras cells grown under permissive culture conditions, iii) YAMC-Ras cells grown under non-permissive culture conditions, iv) IMCE cells, v) IMCE-Ras cells grown under permissive culture conditions, and vi) IMCE-Ras cells grown under non-permissive culture conditions. Colonies were stained with 0.005% w/v crystal violet in PBS containing 4% formaldehyde
  • Figure 9a illustrates the growth of tumours in BALB/c nude mice, wherein BALB/c female nude mice (8 week old) were injected subcutaneously with 0.2 mis of a single cell suspension containing 10 6 cells per site of injection. Tumour growth was assessed twice weekly. Neoplastic masses were measured using calipers. Tumour volume was calculated using the following formula: ) 3 [25]. The mean volumes and standard deviations of 4 tumours are represented, and Figure 9b shows the growth in tumour size over time in animals injected with IMCE-Ras (clone 2) cell line in BALB/c nude mice.
  • Figure 9c shows the results of analysis of tumour tissue for Ras protein expression by means of the Ras autokinase assay. Lanes are as follows: (1) tumour tissue from IMCE-Ras cell line, (2) tumour tissue from YAMC-Ras cell line, and
  • Figure 9d shows the results of analysis of tumour samples for the presence of the APC gene. Lanes are as follows:
  • Heterozygous Immortomice (12) were purchased from Charles River Laboratories (Wilmington, MA. ) .
  • the original Immortomouse strain was generated in CBA/Ca x C57B110 mice, and in the laboratory, the immortalizing gene is carried on a C57B16 background.
  • Min/+ mice (1) were, a gift from Dr Amy Moser, McArdle
  • the small intestine and colon were opened and the contents removed by washing with saline.
  • the culture technique has been described previously (11) .
  • the surface of these tissues was sterilised by soaking the tissues in 0.04% sodium hypochlorite in phosphate-buffered saline (PBS) for 20 minutes.
  • PBS phosphate-buffered saline
  • the tissues were then washed in PBS and incubated for 90 minutes in a solution containing 0.5mM EDTA and 0.05mM dithiothreitol.
  • the incubating solution was then discarded and replaced with PBS, and the container shaken vigorously by hand for 1 minute. This shaking detached the crypts from the underlying stroma.
  • the crypt suspension was placed in a centrifuge tube.
  • the crypt suspensions were cultured in RPMI 1640 medium containing 2.5% fetal calf serum, 10% conditioned medium from the human colonic epithelial carcinoma cell line LIM1863 (14) and 10 units per ml mouse ⁇ -interferon
  • the conditioned medium and ⁇ -interferon are considered to be advantageous but not essential components of the medium.
  • the cells were cultured at a concentration of 300 crypts per well in 24 well plates coated with rat tail collagen. The plates were incubated at the permissive temperature (33°C) in an atmosphere of 5% C0 2 . Fresh ⁇ -interferon was added to the wells every 48 hours for the first 10 days of the culture because the SV40 large T gene incorporated in these cells is coupled to a ⁇ -interferon inducible promoter (12) . A range of 1-100 units per ml is contemplated, but 10 units per ml is preferred. The medium was changed every 4 days until growth was established.
  • fibroblast cultures When confluent, the cultures were split using trypsin/EDTA solution. Pure fibroblast cultures were obtained by trypsinization from the mixed epithelial-fibroblast cultures initially grown from the small intestinal crypts and villi. These fibroblast cultures were grown in medium containing 10% fetal calf serum.
  • the crypts isolated from the colonic mucosa of the Immortomouse/Min hybrid mouse were cultured at the permissive temperature (33°C) in medium containing mouse ⁇ -interferon. Many of the crypts attached to the collagen-coated plates within 24 hours. Unattached crypts were removed and replated into collagen-coated flasks. During the next 14 days the majority of the attached cells died; however, a few cells remained, and these cells proliferated and were passaged when sufficient cells were present. The cultured cells were stored in liquid N 2 before any assays were attempted.
  • the Immortomouse/Min colonic epithelial (IMCE) cells grow as flat cuboidal cells, as shown in Figure 1. The morphology of the cells is similar to the immortomouse-derived colonic epithelial cell line young adult mouse colon (YAMC) described previously (11) . Because the cultures were established from isolated crypts, no stromal cells were found in these cultures.
  • the crypt and villi mixture obtained from the small intestinal mucosa was also cultured.
  • the villi are comprised of both epithelial and stromal cells. These cultures grew slowly, with small areas of epithelial cells being surrounded by a fibroblast monolayer. Pure fibroblast cultures (Immorto/Min fibroblasts; IMF) and pure epithelial cell cultures (Immorto/Min small intestine; ISI) have been isolated from these mixed cultures, and have been stored in liquid N 2 .
  • the cells removed by trypsinization were replated into new flasks and incubated for 1 hour. After this time, the unattached cells were removed and transferred to a fresh flask. This process was repeated hourly for 4 hours. Using this selective adherence technique, it was found that the majority of the fibroblasts in the cell suspension adhered rapidly, with the epithelial cells being enriched in later platings.
  • Muscle and skin fibroblast cell lines were established using the same methods as for liver cells, and using tissue isolated from the same individual animal as before.
  • a gastric cell line consisting of epithelial cells, has also been established in culture.
  • the colonic epithelial cells were cultured on Lab-Tek slide culture chambers (Nunc Inc, Naperville, 111) at 33°C for 3 days. The cells were fixed in cold acetone, and the epithelial nature of the cultured cells was determined by staining with anti-keratin antibodies LE41 (keratin 8), LE61 (keratin 18) and LP2K, (keratin 19) kindly provided by Dr E.B. Lane, CRF Laboratory/ Dundee, Scotland (15) .
  • the cells were also stained using a polyclonal anti-keratin antibody which reacts with most keratin epitopes (Dako Corp., Carpinteria, CA), and were tested for the presence of mucin using monoclonal antibodies specific for the Muc-1 and Muc-2 peptide repeat sequences (16) .
  • the cells cultured from the liver were also tested for the presence of keratin fibres using the same methods.
  • the liver cultures initially contained a mixture of fibroblasts and epithelial cells.
  • An Immorto/Min liver epithelial (IMLE) cell line was obtained by separating the epithelial cells from the stromal cells by differential trypsinisation and differential plating (Fig. 3).
  • the cultured cells showed little staining with the antibody to keratin 18 but did stain with the polyclonal anti-keratin antiserum which recognises most keratin epitopes.
  • the colonic epithelial cells were trypsinised and plated at 2xl0 4 per ml in the wells of 2 -well dishes.
  • the requirement of the cells for ⁇ -interferon was tested by culturing in medium alone or medium containing 10 units/ml of ⁇ -interferon.
  • the conditional immortalization of the cells was tested by culturing duplicate plates at either the permissive temperature (33°C) or the non-permissive temperature (39.5°C) for 6 days. At the end of this incubation period, the cell proliferation in each well was assessed by trypsinizing the adherent cells and counting the total cell number per well. All assays were done in triplicate.
  • Mouse DNA was prepared from 1cm of tail tissue cut from mice greater than 2 weeks of age. In the case of cell lines, the cells were trypsinized and washed twice in PBS. DNA was isolated from both mouse tail tissue and cell lines as follows. Mouse tails or cell pellets were incubated in 750 ⁇ l of homogenizing buffer (50mM Tris-HCl, pH 8; lOOmM EDTA; lOOmM NaCl; 1% SDS; 0.5mg/ml Proteinase K) overnight at 55°C. A volume of 310 ⁇ l of 5M NaCl was added and the mixture centrifuged at 13,000rpm for 10 minutes. The supernatant was transferred to a new tube.
  • homogenizing buffer 50mM Tris-HCl, pH 8; lOOmM EDTA; lOOmM NaCl; 1% SDS; 0.5mg/ml Proteinase K
  • oligonucleotides which respectively hybridize to nucleotides 3234 to 3251 and to nucleotides 2828 to 2845 of the SV40 genome, and which each produce a 424bp PCR product.
  • the oligonucleotides which respectively hybridize to nucleotides 3234 to 3251 and to nucleotides 2828 to 2845 of the SV40 genome, and which each produce a 424bp PCR product.
  • Each PCR reaction contained 150ng DNA, 0.2mM each dNTP , 1.5mM MgCl 2 and 2.5U "Tth plus" DNA polymerase (Biotech International Ltd, Bentley, Western Australia) .
  • 6pM of each primer was used for the SV40 large T reaction.
  • 30pM of each primer was used for the Min reaction.
  • the reactions were overlaid with mineral oil and placed into a thermocycler, as described previously (17). At the completion of the PCR reaction, 16 ⁇ l of the reaction mixture was electrophoresed on a 2% gel and the gel was photographed.
  • the results of the PCR assays for the presence of the Min mutation and SV40 large T gene in the cell lines cultured from the Immortomouse/Min hybrid are shown in Figure 5. It can be seen from lanes 4 and 11 that the colonic epithelial line (IMCE) carries both the Min mutation and SV40 large T gene, whereas the control colonic epithelial line (YAMC) , derived from an Immortomouse, only expresses the SV40 large T gene (lanes 7 and 14) .
  • the liver cell line (IMLE) and the fibroblast line (IMF) derived from the hybrid mouse also carry both genes.
  • Temperature-Sensitivity Cells carrying the temperature-sensitive SV40 large T gene will grow only at the permissive temperature, 33°C, and will die if cultured at 37°C; the rate of cell death is even more rapid at 39.5°C than it is at 37°C.
  • the ability of cell lines carrying this gene and a gene of interest to grow at 39.5°C is compared with the ability to grow at 33°C, in order to determine whether the gene of interest has overcome the temperature-sensitivity of the cells.
  • the ability to grow at the non-permissive temperature is the equivalent of the autonomous growth observed in tumour cells. This therefore provides a general test for identification of oncogenes, and tumour- inducing viruses.
  • Infections were set up as follows: 0.5 ml of the supernatant from the ⁇ 2 Ras cell line was added to the target cells (5 x 10 3 cells per ml in a 5 cm petri dish) together with polybrene (SIGMA) at 4 ⁇ g/ml and incubated for 2 hours at 33°C. Following this incubation, the plates were flooded with 5 mis of complete medium [RPMI 1640 plus 10 "5 M -thioglycerol (ICN Chemicals), 1 unit/ml Insulin (SIGMA),10 "6 M Hydro ⁇ ortisone (SIGMA)] plus 5 units/ml interferon- ⁇ (murine recombinant GIBCO/BRL) , and 10% v/v FCS.
  • SIGMA polybrene
  • the cultures were incubated for a further 48 hours at 33°C in an atmosphere of 5% C0 2 .
  • the infected cells were then harvested and cloned in soft agar for selection at a density of 5 x 10 3 cells/plate.
  • YAMC-Ras and IMCE-Ras cell lines were maintained in complete medium plus 5 units/ml interferon- ⁇ , and 10% v/v FCS at 33°C in an atmosphere of 5% C0 2 .
  • clones were analysed for Ras protein expression by both a Ras autokinase assay and by Western blotting.
  • the auto-kinase assay (22) was performed as follows: cell lysates were prepared using LAU buffer (100 mM NaCl, lOmM Tris-HCl pH 7.5, 2mM EDTA, 0.5% sodium deoxycholate, 1% NP40, lOmM MgCl 2 , 100 units trasylol, and 1 mM PMSF) . After a 30 minute incubation on ice, lysates were spun at 12,000 rpm for 10 minutes. Ras protein was immunoprecipitated using the monoclonal antibody Y13-259 (Furth et al; J. Virol., 1982 ⁇ 3_ 294-304) and protein-G beads .
  • the immunoprecipitated ras protein was then subjected to a kinase assay as follows: beads were washed once in LAU buffer and twice in TMD buffer (50 mM Tris-HCl, pH 7.5, 5 mM MgCl 2 and 1 mM DTT). After the second wash protein-G beads were resuspended in 50 ⁇ l of TMD buffer plus lOmM [ ⁇ - 32 P]GTP/5 mM cold ATP and incubated at 37°C for 30 minutes. The samples were then run on a 15% SDS/PAGE gel, proteins were transferred to immobilon PVDF membrane (Millipore) and phosphorylated bands were visualized by autoradiography.
  • TMD buffer 50 mM Tris-HCl, pH 7.5, 5 mM MgCl 2 and 1 mM DTT. After the second wash protein-G beads were resuspended in 50 ⁇ l of TMD buffer plus lOmM [ ⁇ - 32 P]GTP
  • the blot was then subjected to Western blotting procedure using the Y13-259 antibody after which phosphorylated bands were visualised by ECL (Amersham) .
  • the mAPC genotype of the cell lines was determined as in Example 4. The results of analysis for genotype and for expression of Ras protein are shown in Figure 6.
  • Colony formation in soft agar was observed for both the YAMC and the IMCE cell lines under permissive and non-permissive conditions. Colonies were picked and cell lines expressing Ras , as determined by a Ras auto-kinase assay (22), were designated YAMC-Ras and IMCE-Ras.
  • the morphology of the cell lines is illustrated in Figure 8a. That of the YAMC-Ras cell line was subtly changed compared to the parental YAMC cell line in that the cells were slightly refractile (compare Figure 8a i) and ii)).
  • the IMCE-Ras cell line showed the typical spindle shaped appearance of a transformed cell (compare Figure 8a iii) and iv) ) .
  • the IMCE-Ras cells were refractile, no longer contact-inhibited, and formed foci. Soft agar colonies are illustrated in Figure 8b.
  • tumour cell lines including human tumours
  • the tumourigenicity of the YAMC-Ras and IMCE-Ras cell lines was assessed by injecting 0.2 ml of a single cell suspension containing 10 6 cells per site subcutaneously into nude mice.
  • IMCE-Ras cells had developed tumours with a mean volume of 650 ⁇ 120 mm 3 ( Figure 9a), which continued to express the activated Ras protein ( Figure 9b) ) .
  • Figure 9a mean volume of 650 ⁇ 120 mm 3
  • Figure 9b continued to express the activated Ras protein
  • mice developed tumours by 90 days, with a mean volume of 970 ⁇ 600 mm 3 , which also continued to express the activated Ras protein (Figure 9b) ) .
  • Neither of the parental cell lines induced tumours. Histological studies indicated that all tumours were adenocarcinomas.
  • the presence of the Min mutation in the cultured cells does not confer the property of autonomous growth on these cells. This is shown by the fact that the cells will not proliferate at the non-permissive temperature (39.5°C), at which the SV40 large T protein is inactivated (13) . The role of the SV40 large T protein in inducing growth in these cells was further demonstrated by the decrease in growth rate at the permissive temperature when ⁇ -interferon was omitted from the medium. The fact that the IMCE cell line is only conditionally immortalized suggests that this cell line has retained the phenotype of normal intestinal mucosal cells, which cannot be cultured for long periods in vitro, especially if the cells are from adult animals (8,9).
  • genes known to be implicated in colon cancer such as DCC, p53 or analogues thereof, should induce a malignant transformation in these cells, which in turn are expected to cause the cells to grow autonomously and thus to be able to grow at the non-permissive temperature.
  • the IMCE cell line is a valuable tool for the study of these further events.
  • viruses or oncogenes which may also transform the cell line include, but are not limited to, herpes, Epstein-Barr virus, papillamovirus, myc and xaf.
  • herpes Epstein-Barr virus
  • papillamovirus myc and xaf.
  • the invention makes it possible to study the stages leading to malignant transformation by introducing genetic changes into the normal cells and observing the progression through to tumourigenesis of such cells. This is of importance in humans, as the vast majority of human cancers develop in proliferating, less differentiated cells particularly in epithelial tissues (adenocarcinomas) .
  • Min mouse system Another interesting observation in the Min mouse system is the recent description of a modifier locus that seems to govern the development of tumours in Fl hybrids of Min mice with AKR/J and MA/Myj mouse strains (2,4).
  • the hybrid from which the cells of this invention were derived was on a C57B1/6 background, so that these modifier loci should not apply to the cells of the present invention.
  • the Min mouse has also been found to develop mammary tumours (20), but no hepatic tumours have been described in these mice.
  • the availability of cell lines derived from liver and stroma from the same mouse allows the study of the tissue specific elements that lead to these mice developing small intestinal and colonic tumours but not liver cancers.
  • fibroblast and epithelial cell lines from the same animal can be used to elucidate the role of cell type in cancer development.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Environmental Sciences (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Biodiversity & Conservation Biology (AREA)
  • Animal Husbandry (AREA)
  • Organic Chemistry (AREA)
  • Chemical & Material Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Cell Biology (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Plant Pathology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention concerne des animaux portant un gène d'immortalisation ainsi qu'un ou plusieurs gènes d'intérêts, ainsi que des lignées cellulaires pouvant assurer une croissance à long terme in vitro. Des lignées cellulaires dérivées de l'hybride de souris Min/Immorto F1 portent un allèle Apc défectif et sont immortalisés de manière conditionnelle en vertu d'un expression d'un gros anti-gène T SV40 sensible à la température. Ces lignées cellulaires peuvent en outre être transfectées par d'autres gènes d'intérêt tels que l'oncogène Ras afin de les rendre tumorigènes.
PCT/US1995/007255 1994-06-24 1995-06-07 Nouveaux animaux et nouvelles lignees cellulaires WO1996000285A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU28195/95A AU2819595A (en) 1994-06-24 1995-06-07 Novel animals and cell lines

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
AUPM6471A AUPM647194A0 (en) 1994-06-24 1994-06-24 Novel animals and cell lines
AUPM6471 1994-06-24

Publications (1)

Publication Number Publication Date
WO1996000285A1 true WO1996000285A1 (fr) 1996-01-04

Family

ID=3781040

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1995/007255 WO1996000285A1 (fr) 1994-06-24 1995-06-07 Nouveaux animaux et nouvelles lignees cellulaires

Country Status (2)

Country Link
AU (1) AUPM647194A0 (fr)
WO (1) WO1996000285A1 (fr)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE19740571C1 (de) * 1997-09-15 1999-03-18 Gsf Forschungszentrum Umwelt Verfahren zur Stimulation von T-Zellen mit gewünschter Antigenspezifität
WO2004092220A1 (fr) * 2003-04-14 2004-10-28 Board Of Regents, The University Of Texas System Procedes destines a la production ex vivo sans hybridomes d'anticorps polyclonaux ou monoclonaux et a la generation de populations de cellules immortalisees
EP1243646A3 (fr) * 2001-03-19 2005-03-02 Council of Scientific and Industrial Research Drosophile transgénique quant au gène APC humain APC pour des criblages pharmacologiques
WO2007053637A3 (fr) * 2005-11-01 2007-07-26 Wisconsin Alumni Res Found Mutation du gene de la polypose adenomateuse familiale du rat dans la region humaine hautement mutable

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
CELL, Volume 75, issued 19 November 1993, DIETRICH et al., "Genetic Identification of Mom-1, a Major Modifier Locus Affecting Min-Induced Intestinal Neoplasia in the Mouse", pages 631-639. *
EPITH. CELL BIOL., Volume 3, issued 1994, WHITEHEAD et al., "Derivation of Conditionally Immortalized Cell Lines Containing the Min Mutation from the Normal Colonic Mucosa and Other Tissues of an 'Immortomouse'/Min Hybrid", pages 119-125. *
GENOMICS, Volume 15, issued 1993, LUONGO et al., "Mapping of Multiple Intestinal Neoplasia (Min) to Proximal Chromosome 18 of the Mouse", pages 3-8. *
PROC. NATL. ACAD. SCI. U.S.A., Volume 88, issued June 1991, JAT et al., "Direct Derivation of Conditionally Immortal Cell Lines from an H-2Kb-tsA58 Transgenic Mouse", pages 5096-5100. *
PROC. NATL. ACAD. SCI. U.S.A., Volume 90, issued January 1993, WHITEHEAD et al., "Establishment of Conditionally Immortalized Epithelial Cell Lines from Both Colon and Small Intestine of Adult H-2Kb-tsA58 Transgenic Mouse", pages 587-591. *
SCIENCE, Volume 256, issued 01 May 1992, SU et al., "Multiple Intestinal Neoplasia Caused by a Mutation in the Murine Homolog of the APC Gene", pages 668-670. *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE19740571C1 (de) * 1997-09-15 1999-03-18 Gsf Forschungszentrum Umwelt Verfahren zur Stimulation von T-Zellen mit gewünschter Antigenspezifität
KR19990029816A (ko) * 1997-09-15 1999-04-26 게에스에프-포슝스첸트룸 퓌어 움벨트 운트 게준트하이트 게엠베하 바람직한 항원 특이성을 갖는 t 세포의 자극 방법
US6194205B1 (en) 1997-09-15 2001-02-27 Gsf-Forschungszentrum Fur Umwelt Und Gesundheit Gmbh Method for the stimulation of T cells having a desired antigen specificity
EP1243646A3 (fr) * 2001-03-19 2005-03-02 Council of Scientific and Industrial Research Drosophile transgénique quant au gène APC humain APC pour des criblages pharmacologiques
WO2004092220A1 (fr) * 2003-04-14 2004-10-28 Board Of Regents, The University Of Texas System Procedes destines a la production ex vivo sans hybridomes d'anticorps polyclonaux ou monoclonaux et a la generation de populations de cellules immortalisees
WO2007053637A3 (fr) * 2005-11-01 2007-07-26 Wisconsin Alumni Res Found Mutation du gene de la polypose adenomateuse familiale du rat dans la region humaine hautement mutable

Also Published As

Publication number Publication date
AUPM647194A0 (en) 1994-07-21

Similar Documents

Publication Publication Date Title
Unger et al. In vitro expression of the endothelial phenotype: comparative study of primary isolated cells and cell lines, including the novel cell line HPMEC-ST1. 6R
Li Chinese Academy of Sciences
Oyamada et al. Differential Regulation of Gap Junction Protein (Connexin) Genes during Cardiomyocytic Differentiation of Mouse Embryonic Stem Cellsin Vitro
D’Abaco et al. Synergy between Apc min and an activated ras mutation is sufficient to induce colon carcinomas
Fidanza et al. Double knockout of the ALL-1 gene blocks hematopoietic differentiation in vitro
Lanford et al. Differential ability of a T-antigen transport-defective mutant of simian virus 40 to transform primary and established rodent cells
Proby et al. Spontaneous keratinocyte cell lines representing early and advanced stages of malignant transformation of the epidermis
CN101553564B (zh) 肝星状细胞前体及其分离方法
JP2009195142A (ja) 肝臓卵形細胞の取得方法
Smith et al. Expression of the p53 oncogene in acute myeloblastic leukemia.
Sun et al. The minimal set of genetic alterations required for conversion of primary human fibroblasts to cancer cells in the subrenal capsule assay
JP2008102012A (ja) 癌幹細胞の同定および単離方法
Sevignani et al. Tumorigenic conversion of p53-deficient colon epithelial cells by an activated Ki-ras gene.
Xu et al. Expression and significance of Oct4 in bladder cancer
WO1996000285A1 (fr) Nouveaux animaux et nouvelles lignees cellulaires
Ikematsu et al. In vitro pancreatic ductal cell carcinogenesis
CN110172448B (zh) 一种滑膜肉瘤细胞系hSS-005R及其子代细胞系
JP5954775B2 (ja) ｄｅｎｏｖｏ発癌性膵臓癌細胞株
Warner et al. Establishment and characterization of cMYB-expressing human salivary adenoid cystic carcinoma cell lines (UM-HACC-14, UM-HACC-6) and matching patient-derived xenograft model (UM-PDX-HACC-14)
JP3995248B2 (ja) エプスタインバーウイルス陰性のｎｋ細胞株
JP3730624B2 (ja) ヒト癌原遺伝子を保有する、哺乳動物の癌細胞および形質転換された哺乳動物、ならびにその癌原遺伝子を用いた癌診断用キット
Zhang et al. Melanoma cells appropriate pericyte: endothelial cell crosstalk during perivascular invasion in the brain
Klebe Genetic mapping of a human regulator gene
KR102808052B1 (ko) 불멸화된 마모셋 세포주 및 이의 제조방법
Yaniv et al. Establishment and characterization of a cell line derived from human colon adenocarcinoma (HuCCL-14)

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AU CA JP KR US

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FR GB GR IE IT LU MC NL PT SE

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: CA