getting to the neo
Title: Universal stem cells
United States Patent: 6,986,887
Issued: January 17, 2006
Inventors: Lawman; Patricia (Chipley, FL); Lawman; Michael J. P. (Chipley, FL)
Assignee: Morphogenesis, Inc. (Oldsmar, FL)
Appl. No.: 186231
Filed: June 28, 2002
Abstract
The subject invention pertains to materials and methods for preparing multi-potential stem cells having a pre-selected expression of MHC antigens. Stem cells of the subject invention can be used to generate histocompatible tissues/organs for transplantation. The process of the subject invention comprises the use of targeting vectors capable of gene knockout, insertion of site-specific recombination cassettes, and the replacement of histocompatibility alleles in the stem cell. Novel knockout vectors are used to delete designated regions of one chromosome. Recombination cassette vectors are then used to delete the same region on the second chromosome and deposit a site-specific recombination cassette which can be utilized by replacement vectors for inserting the new MHC genes on the chromosome of the engineered cell. The subject invention also pertains to cells, tissues, and transgenic mammal prepared using the methods and materials of the invention.
.......
"Yeast FLP-FRT Site-Specific Recombination System. The pHRC-DR recombination cassette can contain two FRT site-specific recombination sequences flanking bsd (see FIG. 3). The FRT consists of two inverted 13 bp repeats and an 8 bp spacer (O'Gorman, et al., 1991). Two FRTs from pNEOâGAL plasmid (Stratagene) can be used to flank the bsd gene. Bsd, under the control of the TK promoter can be readily obtained from pMC1bsd.A by digestion with Xho I and Sac II or from pGFP/TKpBSD with Bam HI and Bgl II. Alternatively, heterospecific lox sites could be utilized (Sauer 1996). These variant lox sites, having an altered spacer region are not proficient for Cre-mediated recombination with the canonical 34 bp loxP site, but can recombine with each other. By placing different heterospecific lox sites on different alleles, Cre can catalyze independent DNA recombination events at multiple loci in the same cells. "
....................
"In one embodiment of the methods of the invention, the selectable marker is a positive selectable marker gene, a negative selectable marker gene or both. Positive selection marker genes that can be used with the methods and vectors of the subject invention include green fluorescent protein (gfb), â-galactosidase (â-gal), blasticidin deaminase (bsd), dihydrofolate reductase (dhfr) and neomycin (neo). Negative selection marker genes that can be used with the methods and vectors of the subject invention include diptheria toxin-A (DT-A) and thymidine kinase (TK). Other suitable positive and negative selection marker genes are known in the art and can be used according to the present invention. "
"Claim 1 of 12 Claims
1. An in vitro method of altering the histocompatibility phenotype of a human stem cell, comprising:
a) deleting adjacent HLA-B and HLA-C MHC genes from a first chromosome;
b) replacing HLA-B and HLA-C MHC genes on a second chromosome with a site specific cassette, said cassette comprised within a vector that includes a site specific recombination sequence selected from LoxP and FRT;
c) inserting a transgene harboring a heterologous HLA allele and a homologous sequence matching the site specific recombination sequence of step b); and
d) catalyzing said site specific recombination wherein said transgene becomes incorporated into said second chromosome by site specific recombination to provide a stem cell with altered histocompatibility phenotype. "
LINK:
tinyurl.com/3au4tqSome definitions:
pNEOâGAL plasmid this is the plasmid, binder
(Stratagene) this is the company
bacteriophage Cre-lox
yeast FLP-FRT systems
Recombination, Genetic
Animals
DNA, Fungal [chemistry] [metabolism]
Eukaryotic Cells [physiology]
Gene Targeting
Humans
Integrases [metabolism]
Plasmids
Schizosaccharomyces [genetics] [growth & development]
Transposon Resolvases [metabolism]
0 (DNA, Fungal)
EC 2.7.7.- (Cre recombinase)
EC 2.7.7.- (Integrases)
EC 2.7.7.- (Transposon Resolvases)
TYPE THEM IN SEE WHAT YOU FIND!!!!!!!!************@@@@@@@@+++++++++______
Site-specific recombination systems for the genetic manipulation of eukaryotic genomes.
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Genesis. 2006; 44(10):465-76 (ISSN: 1526-954X)
Thomson JG; Ow DW
Plant Gene Expression Center, USDA, Albany, California 94710, USA.
Site-specific recombination systems, such as the bacteriophage Cre-lox and yeast FLP-FRT systems, have become valuable tools for the rearrangement of DNA in higher eukaryotes. As a first step to expanding the repertoire of recombination tools, we screened recombination systems derived from the resolvase/invertase family for site-specific recombinase activity in the fission yeast Schizosaccharomyces pombe. Here, we report that seven recombination systems, four from the small serine resolvase subfamily (CinH, ParA, Tn1721, and Tn5053) and three from the large serine resolvase subfamily (Bxb1, TP901-1, and U153), can catalyze site-specific deletion in S. pombe. Those from the large serine resolvase subfamily were also capable of site-specific integration and inversion. In all cases, the recombination events were precise. Functional operation of these recombination systems in the fission yeast holds promise that they may be further developed as recombination tools for the site-specific rearrangement of plant and animal genomes.
Subject Headings
Major Subject Heading(s) Minor Subject Heading(s) CAS Registry / EC Numbers
Genome
Recombination, Genetic
Animals
DNA, Fungal [chemistry] [metabolism]
Eukaryotic Cells [physiology]
Gene Targeting
Humans
Integrases [metabolism]
Plasmids
Schizosaccharomyces [genetics] [growth & development]
Transposon Resolvases [metabolism]
0 (DNA, Fungal)
EC 2.7.7.- (Cre recombinase)
EC 2.7.7.- (Integrases)
EC 2.7.7.- (Transposon Resolvases)
PreMedline Identifier: 16981199
www.medscape.com/medline/abstract/16981199GOTTA SEE THIS!
"Research Project: TRANSGENE MANAGEMENT THROUGH SITE-SPECIFIC RECOMBINATION
Location: Plant Gene Expression Center Albany_CA
Title: Site-Specific Recombination Systems for the Genetic Manipulation of Eukaryotic Genomes
Authors
OW, DAVID
THOMSON, JAMES
Submitted to: Genesis
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: August 8, 2006
Publication Date: September 15, 2006
Publisher's URL:
www3.interscience.wiley.com/search/allsearch?mode=viewselected&product=journal&ID=112786688&view_selected.x=102&view_selected.y=8 Reprint URL:
www3.interscience.wiley.com/cgi-bin/fulltext/112786688/PDFSTART Citation: Ow, D.W., Thomson, J.G. 2006. Site-Specific Recombination Systems for the Genetic Manipulation of Eukaryotic Genomes. Genesis 44(10):465-476.
Interpretive Summary: This article describes the demonstration of function of seven prokaryotic site-specific recombination systems in the fission yeast. Four of these are dedicated deletion systems, while the remaining three are integration systems that can also perform inversion and deletions. These new recombination systems represent additional genetic tools for the precise manipulation of eukaryotic genomes.
Technical Abstract: Site-specific recombination systems, such as the bacteriophage Cre-lox and yeast FLP-FRT systems, have become valuable tools for the rearrangement of DNA in higher eukaryotes. As a first step to expanding the repertoire of recombination tools, we screened recombination systems derived from the resolvase/invertase family for site-specific recombinase activity in the fission yeast Schizosaccharomyces pombe. Here, we report that seven recombination systems, four from the small serine resolvase subfamily (CinH, ParA, Tn1721 and Tn5053) and three from the large serine resolvase subfamily (Bxb1, TP901-1 and U153) can catalyze site-specific deletion in S. pombe. Those from the large serine resolvase subfamily were also capable of site-specific integration and inversion. In all cases, the recombination events were precise. Functional operation of these recombination systems in the fission yeast holds promise that they may be further developed as recombination tools for the site-specific rearrangement of plant and animal genomes. "
www.ars.usda.gov/research/publications/publications.htm?seq_no_115=201580@@@@@@@@@@%%%%%%%%%%#########
WHEN THE PROKAYOTE INVADES THE EUKARYOTIC SYSTEM, EVOLUTION IS TAKING PLACE!
CAN YOU UNDERSTAND YET? PLEASE PEOPLE TYPE IN THE GENE NAMES ALL OF THEM......START READING THE PATENTS................THESE TOOLS ...........ARE BEING PATENTED TO CARRY ON THE OPERATION OF TRANSFORMATION.
PROKARYOTES ARE ONE CELLED, WE ARE MULTICELLED, BUT ONE=CELLED ARE BEING INCORPORATED INTO US TO MAKE PROKARYOTES SYMBIOTIC WITH THE HUMAN, ANIMALS, PLANTS.........
GAIA...........ONE EARTH, ONE BODY, ONE INTERACTING BEING...........
PLANTS, ANIMALS, HUMANS.........INTERCHANGING GENES........BUT,,,,,,,,,,,,,,
PROKARYOTES IN EUKARYOTES MAKES EVOLUTION HAPPEN, IT IS NOT EVOLUTION...........
PEOPLE ....................IT IS RECONSTRUCTION, DEVOLVING BACK TO WHERE THE LINK, THAT EVOLUTIONISTS THINK IS THERE, THE PROKARYOTES EVOLVED INTO THE EUKAROTES.
BUT,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,THESE ARE INVENTIONS,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,!!!!!!!!!!!!!
iF YOU CANNOT FIND EVOLUTION PROOF IN FOSSILS, IN THE SEASHELLS ON THE MOUNTAINTOPS, AND THOSE WHO KNOW THIS KNOW EXACTLY WHAT I MEAN,
YOU MUST REALIZE EVOLUTION IS STILLLLLLLLLLLL A THEORY...........YOU CANNOT PROVE IT, ONLY BY RECREATING WHAT YOU THINK IT IS, OR BY RE ESTABLISHING EVENTS THAT MAKE IT APPEAR IT HAPPENED.............YOU CONSTRUCT EVOLUTION...............IT IS CALLED EVOLUTIONARY DEVELOPMENT................... YOU MAKE IT HAPPEN.........................YOU INFER AND THIS IS WHAT INFERANCE DOES TO US. IT LIES TO US, IT IS NOT PROOF!
PROKAYOTES ARE USED IN THE CONSTRUCTION OF THESE INVENTIONS, CREATURES ARE BEING INVENTED,,,,,,,,,WE ARE BEING INVENTED.,,,,,,,,,,,,,,,
KEEP LOOKING FOR THE TOTAL HUMAN INVENTION IN FACT, I SHALL TYPE IT IN......
NEXT,,,,,,,,,,,,,,,,,,
ONE WORD neo NEO PNEO pNEO the plasmid used with
Agrobacterium.
This may not make sense but give me time............
We are not specimens..............we are not to be ethnically, medically experimented on when the experiments of the past created the diseases we have, and not fixing them has to take us back to prokaryotism.............TO RECREATE ALL OF LIFE?
THERE IS AND FOR A LONG TIME NOW BEEN CURES FOR EVERYTHING, BECAUSE ALL DISEASES ARE CAUSED BY GENETIC MANIPULATION, WHETHER IT BE PLANTS, FOR THE PROTEINS WE USE, ANIMALS FOR THE PROTEINS WE USE OR USE FOR THE PROTEINS THAT KEEP US ALIVE.
WHEN NEW PROTEINS ARE INFILTRATED INTO US BY WAY OF YEASTS, BY WAY OF ANIMALS WE EAT, BY WAY FOR VEGGIES WE EAT, FRUIT WE PARTAKE, THE WHOLE EVOLUTIONARY TREE OF LIFE HAS OVERTAKEN THE REAL TREE OF LIFE BECAUSE TRUTH JUST CANNOT BE ACCEPTED, TRUTH IS NEVER EASY, IT IS ALWAYS HARD, BUT THE WIDE AND EASY IS THE FASTEST WAY TOWARD THE SINGULARITY ISN'T IT?
THE BIG PICTURE IS BIG AND IT IS NASTY!!!!!!!!!!!!!!!!!!!!!!!!!!!@@@@@@@@@@@
HUMANS are still the same as when we first started, seems Evolutionists are stuck in the mud.
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