MosSCI-mediated exogenous gene expression is modulated by genomic positioning

Although the Mos1 -mediated single-copy insertion (MosSCI) technique has been widely used to generate stable transgenic Caenorhabditis elegans strains, the link between stability of expression and integration site still needs to be explored. Here, experimental evidence is provided that transgenes are not able to match the level of transcription of their native counterpart, and that insertions at certain locations can result in an external stress-mediated increase in expression. Insertion site ttTi5605 on chromosome II was shown to be a superior location, at least when introducing reproduction related genes. Thus, this study provides a reference for the selection of an optimal site for MosSCI which provides acceptable expression performance whilst minimizing undesirable secondary effects.


Total RNA extraction and real-time quantitative PCR
Total RNA was isolated from 2400 age-synchronised L4 worms by means of the Zymo Quick-RNA kit according to the manufacturer's protocol. cDNA was synthesized from 1000 ng RNA using oligo-dT primer (5′-(T) 20 VN-3′) and M-MLV reverse transcriptase (Promega, UK). qPCR was performed on a QuantStudio 5 Real-Time PCR System (Applied Biosystems, UK) using the SYBR Select Master Mix (Applied Biosystems). Acidic ribosomal subunit protein P1 (rla-1) was utilized as housekeeping gene. The fold changes in gene expression were calculated by applying the 2 −ΔΔCT normalization method.

Reproduction assay
Twelve to 14 age-synchronized L4 nematodes were transferred daily to the same well positions of new 12 well plates until egg laying ceased.
The viable larvae were counted one day after eggs hatched and the daily and total number of offspring determined.

RESULTS
Visual inspection of the insertion sites revealed that no coding sequences were interrupted but two were in close proximity to neighboring genes ( the fluorescent intensities were comparable ( Figure 1D).
Next, the transcription of eft-3 (driven by its native promoter) was measured by real-time quantitative PCR (qPCR) and compared to the transcription of the integrated transgene (gfp driven by the eft-3 promoter). The C. elegans strains were exposed to the environmental carcinogen BaP, which is known to induce toxic responses within the worm, [10] to explore whether external stress selectively alters the dynamics of one of the two eft-3-mediated gene expressions.  Figure 2C). However, the expression for CYP1A1 was 1.7-fold upregulated in worms exposed to 40 μM BaP and 2.1-fold when exposed to 640 μM BaP. The expression level of EPHX increased 1.6-and 1.8-fold in worms exposed to 40 and 640 μM BaP, respectively.
The unc-119 mutant is characterized by a dominant egg laying defect [7,8] and this can be rescued by Cbr-unc-119 during the transgenic process. To determine the extent of phenotype rescue, the cumulative reproduction rate was measured in each strain (Figure 3).
The wild-type (N2) worm, employed as the positive control, produced 227.9 ± 11.7 viable larvae. EG6699, which carries Cbr-unc-119 as an unstable extrachromosomal array, and thus acted as the negative con-

DISCUSSION
A plethora of transgenic techniques has been developed in C. elegans, ranging from multicopy extrachromosomal arrays [11][12][13] over targetspecific, low-copy number, transposon-mediated transgenesis [2,3] to newly developed CRISPR/Cas9-mediated genetic engineering. [14][15][16][17] The choice of promoter is an important aspect in genetic engineering as it controls the strength, stability, and manipulation (if desired) of transgene expression. [18] The insertion site is equally important as the chromosomal location can impact expression. [19,20] The present study characterized three integration locations typically used for  [20] Similar findings were reported in yeast, [21] Drosophila, [22] and human cells. [23] It has been hypothesized that expression strength and the position of genome integration is correlated to global chromatin markers, for example, H3K9me3. [24] Histone modifications regulate the transcriptional processes, [25] which in turn are influenced by the environmental stressors, for example, heat shock and xenobiotics exposure [26,27] such as BaP. [28] In zebrafish larvae, BaP is an DNA methylation modifier which controls gene-specific transcriptional adaptation caused by environmental stress. [29] Therefore, the observed elevated transcription in a worm genome modified at the insertion site ttTi5605 (II) and challenged with BaP may have been due to histone modifications. Values represent mean ± standard error of mean (SEM), n = 3 (three biological repeats, each measured three times). Cycle threshold (Ct) and relative expression of human cytochrome P450 Family 1 Subfamily A Member 1 (CYP1A1) and epoxide hydrolase (EPHX) driven by the worm promoter eft-3 compared to eft-3 (with its native promotor) in worms exposed to 0, 40, or 640 μM BaP from age-synchronized L1 to L4 stage. All conditions contained 0.5% DMSO. (C) Ct values of the target genes in humanized CYP1A1_EPHX worms (mean, minimum, and maximum values). (D) Relative expression of target genes in humanized CYP1A1_EPHX worms (mean ± standard error of mean [SEM], n = 9). Statistical analysis (comparing unexposed to exposed) was performed via a two-way analysis of variance (ANOVA), followed by a Tukey's multiple comparison test. ****p < 0.0001, ***p < 0.001.
Others have argued that the variation in expression between the transgene and the analogous native promoter donor gene might be the result of RNAi-like mechanisms. [20] The observation in the present study that expression of the transgene GFP under the eft-3 promoter was lower than the eft-3 under its own (native) promoter supports the hypothesis that transcription can be silenced by small RNAs.
The standard transgenic process requires the C. briggsae unc-119 gene as a positive selection marker of the successful incorporation of the construct, [7] thereby rescuing a dominant deficit in reproductive performance of the unc-119 mutant background. [2] The current work demonstrated that the location and copy-number of Cb-unc-119 insertion impacted the rescue efficiency. The insertion of the transgenic cassette at cxTi10816 (IV) disrupted the 3′ UTR of srm-1, a gene which encodes a serpentine receptor expressed in ganglia and pharynx. Although little is known about the function of this receptor, the disruption of its 3′ UTR (the location determining changes in post-transcriptional regulation [30] might have interfered with the translation of the SRM-1, in turn resulting in the smaller brood size of EG6401. The insertion site ttTi4348 (I) is located within a heavily methylated area (left arm), [31] which may lower the rescue efficiency. Interestingly, the integration site (ttTi5605) in EG6070 displays the highest rescue efficiency, which also coincides with the highest insertion frequency, [3] and possibly an interconnected correlation.
Overall, this study provides experimental evidence that transcrip- the choice of integration site should not be assumed neutral a priori, but rather selected via an empirical-based decision process.

CONFLICT OF INTEREST STATEMENT
The author(s) declare no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. The manuscript contains experiments using invertebrate only; no vertebrates /human studies are included.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.