Engineered landing pads for gene targeting in plants
A method for producing a transgenic plant includes providing a nucleic acid molecule comprising at least two regions of nucleic acid sequence that lack sequence homology with genomic DNA of the plant cell, and at least two zinc finger nuclease recognition sites, wherein the at least two regions of nucleic acid sequence that lack sequence homology with genomic DNA of the plant cell flank the at least two zinc finger nuclease recognition sites. A plant cell or tissue having the nucleic acid molecule stably integrated into the genome of the plant cell is transformed. A plant is regenerated from the plant cell. Transgenic plants are produced by the method. Seeds are produced by the transgenic plants.
1. A method for producing a transgenic plant cell, the method comprising:
identifying a nucleotide sequence not comprised within the genomic DNA of the plant cell;
transforming the plant cell with a nucleic acid molecule comprising a polynucleotide that comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs:15, 16 and 19 and a targeting endonuclease recognition site,
such that the polynucleotide is stably integrated into the genome of the plant cell at a genomic location,
thereby producing a transgenic plant cell comprising a unique site for targeted genetic modification defined by the genomic location of the polynucleotide.
2. The method according to claim 1 , wherein the polynucleotide further comprises a first restriction site positioned 5′ of the nucleotide sequence that is recognized by a first restriction enzyme, and a second restriction site positioned 3′ of the nucleotide sequence that is recognized by a second restriction enzyme.
3. The method according to claim 2 , wherein the first and second restriction sites generate compatible single-stranded ends when subjected to restriction digestion, and wherein hybrid ligated sites formed by ligation of the compatible single-stranded ends are not recognized by either of the first and second restriction enzymes.
4. The method according to claim 1 , wherein the nucleotide sequence are each between 50 bp and 3 kb in length.
5. The method according to claim 1 , wherein the targeting endonuclease recognition site is a recognition site for a zinc finger nuclease.
6. The method according to claim 1 , the method further comprising:
introducing into the transgenic plant cell a targeting endonuclease that recognizes the targeting endonuclease recognition site, and a second nucleic acid molecule comprising a second nucleotide sequence and a third nucleotide sequence flanking a heterologous polynucleotide of interest in the second nucleic acid molecule, wherein the third nucleotide sequence is homologous to the first nucleotide sequence, and wherein the second nucleotide sequence is homologous to the first nucleotide sequence, such that the heterologous polynucleotide of interest is stably integrated into the genome of the transgenic plant cell by homologous recombination within the first nucleotide sequence, thereby producing a transgenic plant cell comprising the heterologous polynucleotide integrated at the unique site for targeted genetic modification.
7. The method according to claim 6 , wherein the targeting endonuclease is a zinc finger nuclease.
8. The method according to claim 6 , wherein the transgenic plant cell is comprised in a transgenic plant tissue, transgenic plant part, or whole, transgenic plant.
9. The method according to claim 6 , further comprising regenerating a transgenic plant from the transgenic plant cell.
10. The method of claim 6 , wherein the targeting endonuclease is introduced into the transgenic plant cell by transforming the plant cell with a polynucleotide encoding the targeting endonuclease.
11. The method according to claim 6 , wherein the targeting endonuclease is introduced into the transgenic plant cell by crossing a transgenic plant comprising the second nucleic acid molecule with a transgenic plant comprising the targeting endonuclease.
12. The method according to claim 6 , wherein the second nucleic acid molecule is introduced into the transgenic plant cell by crossing a transgenic plant comprising the targeting endonuclease with a transgenic plant comprising the second nucleic acid molecule.
13. The method according to claim 6 , wherein the targeting endonuclease and the second nucleic acid molecule are introduced into the transgenic plant cell by crossing a transgenic plant comprising the transgenic plant cell with a transgenic plant comprising the targeting endonuclease and the second nucleic acid molecule.
14. The method according to claim 6 , wherein the second nucleic acid molecule further comprises a first restriction site positioned 5′ of the third and fourth nucleotide sequences that is recognized by a first restriction enzyme, and a second restriction site positioned 3′ of the third and fourth nucleotide sequences that is recognized by a second restriction enzyme.
15. The method according to claim 14 , further comprising introducing into the transgenic plant cell at least one endonuclease that recognizes the first and second restriction sites, such that the heterologous polynucleotide of interest is excised from the genome of the plant cell.