Methods and systems for predicting the risk of transgene silencing

Abstract

The present invention is based on the use of the H3K9me2 methylation levels in plant genomes to predict transgene silencing, transgene stability, and/or transgene expression level. Provided are methods and/or systems for generating whole-genome H3K9me2 maps and its use with an assigned threshold value for predicting gene silencing. The methods and/or systems provided herein can be used in high-throughput setting for screening large number of transformed event in a relatively short period of time as compared to existing technologies.

What is claimed is: 
     
         1 .- 15 . (canceled) 
     
     
         16 . A computerized system for predicting the risk of transgene silencing, comprising,
 (a) a histone methylation/acetylation database showing peak numbers of the histone methylation/acetylation profiles in a plant's genome;   (b) a first computer module for the input of data where a user inputs sequences of at least one transgene insertion site from a transgenic event; and   (c) a second computer module for the output of data where data predicting the risk of transgene silencing is generated to select a transgenic event with a lower probability of transgene silencing, wherein the data predicting the risk of transgene silencing is based on comparison of the inputted sequences of Step (b) and the histone methylation/acetylation profiles of Step (a).   
     
     
         17 . The system of  claim 16 , wherein the histone methylation/acetylation profile data using a chromatin immunoprecipitation sequencing (ChIP-seq) assay. 
     
     
         18 . The system of  claim 16 , wherein the histone methylation/acetylation profile data is associated with histone methylation/acetylation selected from the group consisting of H3K4me2, H3K4me3, H3K9/14ac, H3K9me2, H3K9me3, H3K27me1, H3K27me3 and H4K20me3, and combinations thereof. 
     
     
         19 . The system of  claim 16 , wherein the histone methylation/acetylation profile data is associated with histone methylation/acetylation selected from the group consisting of H3K9me2, H3K9me3, H3K27me1, H3K27me3 and H4K20me3, and combinations thereof. 
     
     
         20 . The system of  claim 16 , wherein the histone methylation/acetylation profile data is associated with H3K9me2 methylation. 
     
     
         21 . The system of  claim 16 , wherein the plant is selected from soybean, maize, canola, cotton, wheat, and rice. 
     
     
         22 . A process for use in a computerized system for predicting the risk of transgene silencing, comprising,
 (a) inputting sequences of at least one transgene insertion site into the system of  claim 16  by a user; and   (b) receiving output from the system of  claim 16  for prediction of the risk of transgene silencing, wherein the prediction is based on comparison of the inputted sequences of the transgene insertion site and the histone methylation/acetylation profiles within the system.   
     
     
         23 . A transgenic plant, plant part, or plant cell comprising a transgene inserted within a genomic locus, wherein the transgene insertion is calculated to fall into peak zero or peak one of the histone methylation/acetylation profile as determined by the method comprising,
 a. generating histone methylation/acetylation profile data of a plant's genome from a physical assay;   b. assembling the histone methylation/acetylation profile data of step (a) into a histone methylation/acetylation database showing peak numbers of the histone methylation/acetylation profiles;   c. analyzing sequences of at least one transgene insertion site from a transgenic event;   d. comparing the sequences of the transgene insertion site of step (c) with the histone methylation/acetylation database of step (b); wherein
 i. if the transgene insertion site falls into peak zero or peak one of the histone methylation/acetylation profiles, there is no risk of transgene silencing, or 
 ii. if the transgene insertion site falls into peak two or more of the histone methylation/acetylation profiles, there is significant risk of transgene silencing; and, 
   e. selecting the transgenic event with a lower probability of transgene silencing.   
     
     
         24 . The transgenic plant of  claim 23 , wherein the histone methylation/acetylation profile data is generated using a chromatin immunoprecipitation sequencing (ChIP-seq) assay. 
     
     
         25 . The transgenic plant of  claim 23 , wherein the histone methylation/acetylation profile data is associated with histone methylation/acetylation selected from the group consisting of H3K4me2, H3K4me3, H3K9/14ac, H3K9me2, H3K9me3, H3K27me1, H3K27me3 and H4K20me3, and combinations thereof. 
     
     
         26 . The transgenic plant of  claim 23 , wherein the histone methylation/acetylation profile data is associated with histone methylation/acetylation selected from the group consisting of H3K9me2, H3K9me3, H3K27me1, H3K27me3 and H4K20me3, and combinations thereof. 
     
     
         27 . The transgenic plant of  claim 23 , wherein the histone methylation/acetylation profile data is associated with H3K9me2 methylation. 
     
     
         28 . The transgenic plant of  claim 23 , wherein the transgenic plant is selected from soybean, maize, canola, cotton, wheat, and rice. 
     
     
         29 . The transgenic plant of  claim 23 , wherein the sequences of the transgene insertion site are obtained by sequencing.