Methods of repairing tandemly repeated DNA sequences and extending cell life-span using nuclear transfer
This invention relates to methods for rejuvenating normal somatic cells and for making normal somatic cells of a different type having the same genotype as a normal somatic cell of interest. These cells have particular application in cell and tissue transplantation. Also encompassed are methods of re-cloning cloned animals, particularly methods where the offspring of cloned mammals are designed to be genetically altered in comparison to their cloned parent, e.g., that are “hyper-young.” These animals should be healthier and possess desirable properties relative to their cloned parent. Also included are methods for activating endogenous telomerase, EPC-1 activity, and or the ALT pathway and/or extending the life-span of a normal somatic cell, and other genes associated with cell aging and proliferation capacity.
1 . A method comprising:
a. transferring a first cell, the nucleus from said first cell or chromosomes from a first cell to a recipient oocyte or egg in order to generate an embryo;
b. obtaining an inner cell mass, embryonic disc and/or stem cell using said embryo;
c. injecting said inner cell mass, embryonic disc and/or stem cell into an immune-compromised animal to form a teratoma;
d. isolating said resulting teratoma;
e. isolating a second cell from said teratoma, wherein said second cell is of a desired type.
2 . The method of claim 1 , wherein said first cell is a senescent cell or a cell that is near senescence.
3 . The method of claim 1 , wherein said cell isolated from said nuclear transfer teratoma has telomeres that are on average at least as long as or longer than those of cells from a same age control teratoma that is not generated by nuclear transfer techniques.
4 . (canceled)
5 . The method of claim 2 , wherein said first cell is a fibroblast.
6 . The method of claim 1 , wherein said immune-compromised animal is a SCID or nude mouse.
7 . The method of claim 1 , wherein said first cell has at least one genetic alteration.
8 . The method of claim 1 , wherein a said second cell is of a different type than the first cell.
9 . (canceled)
10 . (canceled)
11 . The method of claim 1 , wherein said second cell is of a type selected from the group consisting of smooth muscle, skeletal muscle, cardiac muscle, skin and kidney.
12 . The method of claim 1 , further comprising growing said cell of a different type in the presence of growth factors to facilitate further differentiation.
13 . (canceled)
14 . (canceled)
15 . The cell isolated by the method of claim 1 or a tissue comprising said cell.
16 . (canceled)
17 . The method of claim 7 , wherein said genetic alteration comprises the transfection of at least one heterologous gene or the disruption of at least one native gene.
18 - 24 . (canceled)
25 . The method of claim 21 , further comprising:
a. transferring the nucleus of said second cell into a recipient oocyte,
b. generating an embryo or embryonic stem cell from said recipient oocyte,
c. introducing said embryo or embryonic stem cell into a recipient female, and
d. allowing said embryo or embryonic stem cell to fully develop such that said female delivers a newborn animal having the same genotype as said primary cell, wherein said newborn animal is non-human.
26 . (canceled)
27 . (canceled)
28 . (canceled)
29 . The method of claim 1 , further comprising:
f. prior to step (a), making a first genetic modification to said first cell by inserting heterologous DNA and/or deleting native DNA,
g. prior to step (a), allowing said genetically modified primary cell to multiply to senescence or near-senescence,
h. making a second genetic modification to said second cell by inserting heterologous DNA and/or deleting native DNA,
i. allowing the cell produced in step (h) to multiply until senescence or near senescence,
j. using the senescent or near-senescent cell produced in step (i) as a nuclear donor for nuclear transfer to an enucleated oocyte or an enucleated fertilized egg, and
k. obtaining a re-cloned inner cell mass, blastocyst, teratoma, embryo, fetus or animal having said first and second genetic modifications.
30 . The method of claim 29 further comprising steps where said re-cloned inner cell mass, blastocyst, teratoma, embryo, fetus or animal is again re-cloned, thereby producing a further re-clone, and wherein a third genetic modification is made such that the further re-clone has the first, second and third genetic modifications.
31 . The method of claim 30 , wherein said further re-clone is generated by nuclear transfer using a senescent or near-senescent donor cell.
32 . The method of claim 29 , wherein said further re-clone has telomeres that are at least as long on average as a same age control animal that was not generated using nuclear transfer techniques.
33 . The method of claim 31 , wherein said further re-clone has telomeres that are at least as long on average as a same age control animal that was not generated using nuclear transfer techniques.
34 . The method Of claim 29 , wherein the genetic modifications involve genes that are responsible for immunological function.
35 - 39 . (canceled)
40 . A method of identifying at least one gene or protein that either directly or indirectly enhances or decreases telomerase activity, comprising screening a cDNA or mRNA library generated from an embryo or embryonic stem cell, or screening a fraction from an oocyte or embryonic stem cell, for members that enhance or decrease telomerase activity in a senescent or near-senescent cell.
41 - 70 . (canceled)
71 . The method of claim 1 , wherein said first cell is of a species selected from the group consisting of human, bovine, ungulate, equine, canine, feline, porcine, mouse, rat, goat, sheep, guinea pig, bear, rabbit.
72 - 86 . (canceled)