Artificial chromosomes, uses thereof and methods for preparing artificial chromosomes
View Patent ↗Methods for amplification of nucleic acids in cells are provided. Also provided are cells that contain the nucleic acids.
1. A method for producing a cell that contains heterologous nucleic acid, comprising introducing a satellite artificial chromosome (SATAC) into a plant cell, wherein:
a SATAC is an artificial chromosome that contains more heterochromatin than euchromatin;
the cell is a plant cell; and
the SATAC that is introduced is prepared by a method, comprising:
introducing a DNA fragment into a plant cell, wherein the DNA fragment comprises a selectable marker and a targeting sequence of nucleotides that comprises rDNA that targets the fragment to the pericentric heterochromatic region of a chromosome in the cell;
growing the cell under selective conditions to produce cells that have incorporated the DNA fragment or a portion thereof that comprises the selectable marker into their genomic DNA in the pericentric heterochromatin of a chromosome in the cell, resulting in amplification of the region containing pericentric heterochromatin resulting in duplication of a centromere derived from the plant cell and producing a dicentric chromosome, which fragments and undergoes further amplification to produce a satellite artificial chromosome (SATAC); and
identifying and selecting a cell that comprises a chromosome that contains the amplified pericentric heterochromatin and the DNA fragment or portion thereof comprising the selectable marker, wherein the chromosome is a satellite artificial chromosome (SATAC).
2. The method of claim 1 , wherein the SATAC is introduced by a method selected from among direct DNA transfer or uptake, electroporation, lipofection, nuclear microinjection, liposome mediated transfer, microprojectile bombardment, polycation-mediated transfer and microcell fusion.
3. The method of claim 1 , wherein the SATAC is introduced by a method selected from among microinjection, nuclear transfer, electrofusion, projectile bombardment and lipid-mediated transfer systems.
4. The method of claim 1 , wherein the SATAC that is introduced is an isolated substantially pure SATAC.
5. The method of claim 1 , wherein the SATAC is a megachromosome, comprising about 50 to about 450 megabases (Mb).
6. The method of claim 1 , wherein the SATAC is a megachromosome, comprising about 250 to about 400 Mb.
7. The method of claim 1 , wherein the SATAC is a megachromosome, comprising about 150 to about 200 Mb.
8. The method of claim 1 , wherein the SATAC is a megachromosome, comprising about 90 to about 120 Mb.
9. The method of claim 1 , wherein the SATAC is a megachromosome, comprising about 15 to about 60 Mb.
10. The method of claim 1 , wherein the plant is tobacco, rice, maize, rye, soybean, Brassica napus , cotton, lettuce, potato, tomato or arabidopsis.
11. The method of claim 1 , wherein the SATAC comprises nucleic acid encoding a gene product or a plurality of gene products.
12. The method of claim 11 , wherein the gene products comprise a biosynthetic pathway.
13. The method of claim 1 , wherein the plant cell is a protoplast.
14. The method of claim 1 , wherein the SATAC is introduced into the plant cell by cell fusion or microcell fusion.
15. A method for producing a cell that contains heterologous nucleic acid, comprising:
introducing a satellite artificial chromosome (SATAC) into a cell, wherein:
a SATAC is an artificial chromosome that contains more heterochromatin than euchromatin;
the cell is a plant cell;
the SATAC is introduced into the plant cell by a method, comprising:
introducing a DNA fragment into the plant cell, wherein the DNA fragment comprises a selectable marker and a targeting sequence of nucleotides that comprises rDNA that targets the fragment to the pericentric heterochromatic region of a chromosome in the cell; and
growing the cell under selective conditions to produce cells that have incorporated the DNA fragment or a portion thereof that comprises the selectable marker into their genomic DNA in the pericentric heterochromatin of a chromosome in the cell, wherein incorporation results in amplification of the region containing pericentric heterochromatin resulting in duplication of a centromere derived from the plant cell and producing a dicentric chromosome, which fragments and undergoes further amplification to produce a satellite artificial chromosome (SATAC); and
identifying and selecting a cell that comprises a chromosome that contains the amplified pericentric heterochromatin and the DNA fragment or portion thereof comprising the selectable marker, wherein the chromosome is a satellite artificial chromosome (SATAC), thereby introducing a SATAC into the cell.
16. The method of claim 15 , further comprising isolating the SATAC from the cell in which the SATAC is produced.
17. A method for producing a cell that
contains heterologous nucleic acid, comprising:
introducing a DNA fragment into a plant cell, wherein the DNA fragment comprises a selectable marker and a targeting sequence of nucleotides that comprises rDNA that targets the fragment to the pericentric heterochromafic region of a chromosome;
growing the cell under selective conditions to produce cells that have incorporated the DNA fragment or a portion thereof that comprises the selectable marker into their genomic DNA in the pericentric heterochromatin of a chromosome in the cell, wherein incorporation results in amplification in the region containing pericentric heterochromatin resulting in duplication of a centromere derived from the plant cell and producing a dicentric chromosome, which fragments and undergoes further amplification to produce a sausage chromosome containing amplified heterochromatin and the DNA fragment or portion thereof amplified and interspersed in the heterochromatin; and
selecting and isolating a cell that comprises a sausage chromosome that contains the amplified pericentric heterochromatin and the DNA fragment or portion thereof comprising the selectable marker.
18. The method of claim 17 , wherein the plant cell is a protoplast.
19. A method for producing a cell that
contains heterologous nucleic acid, comprising introducing a satellite artificial chromosome (SATAC) into a cell, wherein:
a SATAC is an artificial chromosome that contains more heterochromatin than euchromatin;
the cell is a plant cell; and
the SATAC that is introduced into the plant cell is produced by a method, comprising:
introducing a DNA fragment into a cell, wherein the DNA fragment comprises a selectable marker and a targeting sequence of nucleotides that comprises rDNA that targets the fragment to the pericentric heterochromatic region of a chromosome;
growing the cell under selective conditions to produce cells that have incorporated the DNA fragment or a portion thereof that comprises the selectable marker into their genomic DNA in the pericentric heterochromatin of a chromosome in the cell, wherein incorporation results in amplification of the region containing pericentric heterochromatin resulting in duplication of a centromere derived from the plant cell and producing a dicentric chromosome, which fragments and undergoes further amplification to produce a satellite artificial chromosome (SATAC); and
identifying and selecting a cell that comprises a chromosome that contains amplified pericentric heterochromatin and the DNA fragment or portion thereof comprising the selectable marker; and
isolating a cell that comprises the SATAC.