Uniform Expression and Relatively Small Position Effects Characterize Sister Transformants in Maize and Soybean.

Scott D Betts,Dave Peterson,Russ Booth,Jeffry D Sander,Dave Stucker,Sutirtha Basu,Xiaoyi Sopko,Zhongsen Li,Jeffry Farrell,Huirong Gao,Brian Lenderts,N Doane Chilcoat,A Mark Cigan,Shujun Chang,Chris Scelonge,Emily Wu,Michelle Mcenany,Joy Bolar,Anthony J Kinney ,Jasdeep S Mutti ,Ling Lü ,Kristi R Harkins

doi:10.3389/fpls.2019.01209

Abstract

Development of transgenic cell lines or organisms for industrial, agricultural, or medicinal applications involves inserting DNA into the target genome in a way that achieves efficacious transgene expression without a deleterious impact on fitness. The genomic insertion site is widely recognized as an important determinant of success. However, the effect of chromosomal location on transgene expression and fitness has not been systematically investigated in plants. Here we evaluate the importance of transgene insertion site in maize and soybean using both random and site-specific transgene integration. We have compared the relative contribution of genomic location on transgene expression levels with other factors, including cis-regulatory elements, neighboring transgenes, genetic background, and zygosity. As expected, cis-regulatory elements and the presence/absence of nearby transgene neighbors can impact transgene expression. Surprisingly, we determined not only that genomic location had the least impact on transgene expression compared to the other factors that were investigated but that the majority of insertion sites recovered supported transgene expression levels that were statistically not distinguishable. All 68 genomic sites evaluated were capable of supporting high-level transgene expression, which was also consistent across generations. Furthermore, multilocation field evaluation detected no to little decrease in agronomic performance as a result of transgene insertion at the vast majority of sites we evaluated with a single construct in five maize hybrid backgrounds.

Highlights

The development of genetically modified organisms or cell lines for commercial purposes involves inserting DNA sequences into the nuclear genome to generate a trait of interest
(Zastrow-Hayes et al, 2015), we evaluated transformants generated by Agrobacterium transformation and by site-specific integration (SSI) and identified individual events that contained only the intended DNA insertion. quantitative PCR (qPCR) was used to determine the copy number of transgenic sequences, whereas SbS used a combination of oligo-based target enrichment and Illumina-based sequencing to determine the presence and location of transgenic DNA
A central paradigm in transgenic biology is that chromosomal location, that is, position effect, is an important determinant of transgene expression

Summary

Introduction

The development of genetically modified organisms or cell lines for commercial purposes involves inserting DNA sequences into the nuclear genome to generate a trait of interest. It has been observed that independent sister transformants, that is, transgenic cells/organisms generated using the same DNA construct and containing one or more DNA insertions or “events” can exhibit dramatically different transgene expression levels (Strauss and Sax, 2016) This wide variation in transgene expression among sister transformants has led to the inference that genomic insertion site plays a central role in transgene expression (Matzke and Matzke, 1998; Cocciolone et al, 2000; Cantos et al, 2014) and has led to a product development paradigm wherein large numbers of sister transformants are generated and phenotyped to select a single transgenic event that has an expression level leading to appropriate trait efficacy in elite hybrids or varieties suitable for commercialization (Sachs et al, 1998; Bradford et al, 2005; Burns et al, 2015). Position effects have been attributed to higher order chromatin structural variation

Methods

Results

Conclusion