Background

Quantifying specific spatial or temporal expression of a transgenically expressed endogenous gene against a background of high or overlapping expression of the native endogenous sequence remains a long-standing problem in eukaryotic molecular research. To overcome this, we have developed a protocol based on the introduction of a synonymous mutation into the transgenic copy of the endogenous gene to remove a restriction enzyme cleavage site. This is frequently a feature of the assembly of Golden Gate Cloning constructs (the so-called domestication process, involving excision of key Type IIS restriction nuclease sites) (Engler et al., 2008; Weber et al., 2011). This restriction site difference can then be exploited to differentiate between endogenous and transgenic expression by digesting complementary DNA (cDNA) from transgenic individuals with the relevant restriction enzyme, which will cleave endogenous cDNA but will leave the transgene cDNA uncut. Misexpression of an endogenous gene in a tissue where it is not normally expressed using a cell type–specific promoter is a commonly used experimental technique (Prelich, 2012). However, if that cell type is temporally or spatially difficult to isolate, it can be challenging to determine accurately the levels of overexpression that have been achieved without technically complex and laborious dissection. The protocol removes the need to isolate the specific cell types to confirm expression but instead allows this to be done on RNA extracted from bulk tissues.

Essentially, the protocol set out below involves making a domesticated version of the transgene missing a restriction site present in the endogenous sequence and transforming it into the organism under study. Following RNA extraction and standard reverse transcription, an additional digestion step is introduced whereby the cDNA is restricted using the enzyme targeting the site now only present in cDNA from the endogenous gene. Subsequent qPCR, using primers amplifying a region of the gene containing the restriction site, will thus only amplify cDNA specific to the transgene, leaving the endogenous cDNA unaffected—since it can no longer serve as a template for amplification. qPCR on this digested cDNA using primers amplifying a region of a housekeeping gene that does not contain the restriction site allows the calculation of relative expression levels using the 2^-ΔΔC(T) method (Livak and Schmittgen, 2001). Although this protocol is simplest to implement in cases where Golden Gate cloning is used and an endogenous Bpi1 restriction enzyme cut site has been removed, with planning during cloning this same principle can also be adapted to any other cloning strategy by the introduction of a synonymous mutation that removes a restriction enzyme cleavage site from the transgene. Overall, this technique allows very rapid and accurate estimation of the transgene expression level, as negligible endogenous cDNA survives cleavage and thus expression levels detected by qPCR reflect only the transgene. cDNA from wildtype organisms can, of course, be used to determine approximate control levels of endogenous gene expression and, in the case of inducible transgenes, a more accurate estimate of endogenous gene expression can be obtained by sampling cDNA from uninduced tissue.

This method was developed by Bezodis et al. (2022) to detect expression from inducible constructs that ectopically express high levels of homeodomain transcription factors in a spatially and temporally restricted region of the germline lineage of the model plant Arabidopsis thaliana. While the transcription factors of interest are normally not expressed in the germline, they are expressed significantly throughout the surrounding somatic tissues. These endogenous transcripts thus constitute a major component of the RNA collected from whole tissues but, by digesting cDNA from the endogenous gene copy carrying the functional restriction site, this method avoids the laborious task of dissecting out cells in which only the transgene is expressed. The combination of this qPCR strategy and the use of imaging markers has permitted both the location and extent of transgene expression to be accurately determined using promoters driving expression in specific germline cell types over a short period. We have found the use of an inducible system to be particularly helpful, as it provides a control expression level to confirm that the digest has worked effectively at removing expression of the endogenous gene and matching construct-driven expression to observed phenotypes (Bezodis et al., 2022). Induction uses a two-component LhGR/pOp6 expression system (Craft et al., 2005) and a protocol for this system has been published (Samalova et al., 2019), including for use in reproductive tissues (Schubert et al., 2022). We note, however, that the protocol does not require an inducible system, nor is it limited to Arabidopsis or, in fact, to plants.