# Potential genetic robustness of Prnp and Sprn double knockout mouse embryos towards ShRNA-lentiviral inoculation

Andrea Rau, Bruno Passet, Johan Castille, Nathalie Daniel-Carlier, Alexandre Asset, Jérome Lecardonnel, Marco Moroldo, Florence Jaffrézic, Denis Laloë, Katayoun Moazami-Goudarzi, et al.

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## To cite this version:

Andrea Rau, Bruno Passet, Johan Castille, Nathalie Daniel-Carlier, Alexandre Asset, et al. Potential genetic robustness of Prnp and Sprn double knockout mouse embryos towards ShRNA-lentiviral inoculation. Veterinary Research, 2022, 53, 10.1186/s13567-022-01075-4. hal-03718138

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Submitted on 8 Jul 2022

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SHORT REPORT

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# Potential genetic robustness of *Prnp* and *Sprn* double knockout mouse embryos towards ShRNA-lentiviral inoculation

Andrea Rau<sup>1,2* ID</sup>, Bruno Passet<sup>1</sup>, Johan Castille<sup>1</sup>, Nathalie Daniel-Carlier<sup>1</sup>, Alexandre Asset<sup>1</sup>, Jérôme Lecardonnel<sup>1</sup>, Marco Moroldo<sup>1</sup>, Florence Jaffrézic<sup>1</sup>, Denis Laloë<sup>1</sup>, Katayoun Moazami-Goudarzi<sup>1</sup> and Jean-Luc Vilotte<sup>1</sup>

**Abstract**

The Shadoo and PrP prion protein family members are thought to be functionally related, but previous knockdown/ knockout experiments in early mouse embryogenesis have provided seemingly contradictory results. In particular, Shadoo was found to be indispensable in the absence of PrP in knockdown analyses, but a double-knockout of the two had little phenotypic impact. We investigated this apparent discrepancy by comparing transcriptomes of WT, *Prnp*<sup>0/0</sup> and *Prnp*<sup>0/0</sup>*Sprn*<sup>0/0</sup> E6.5 mouse embryos following inoculation by *Sprn*<sup>-</sup> or *Prnp*-ShRNA lentiviral vectors. Our results suggest the possibility of genetic adaptation in *Prnp*<sup>0/0</sup>*Sprn*<sup>0/0</sup> mice, thus providing a potential explanation for their previously observed resilience.

**Keywords:** Mouse, prion, Shadoo, lentivirus, ShRNA, robustness

**Introduction**

The prion protein PrP, encoded by *Prnp*, is strongly associated with several neurodegenerative diseases; in particular, misfolded isoforms of PrP are thought to be a key component of the infectious prions that cause Transmissible Spongiform Encephalopathy. PrP is evolutionarily related to another member of the prion protein family, Shadoo, which is encoded by *Sprn* [1]. However, their individual biological functions and the complex inter-relationship between the two remain poorly characterized. Previous single and double knockdown experiments in early mouse embryogenesis have provided seemingly contradictory results. Individual genetic invalidations yielded little phenotypic impact beyond resistance to prion infection for *Prnp*-knockout mice. Knockdown of

*Sprn* in *Prnp*-knockout embryos was found to induce early embryonic lethality as early as E7.5 linked to the developmental failure of the trophectoderm-derived compartment [2]. Although these results together appeared to suggest a potential biological redundancy of the two proteins, double genetic invalidations of *Prnp* and *Sprn* in mice with various genetic backgrounds [3, 4] did not confirm this hypothesis; we note that all experiments involved FVB/N genetic backgrounds, obtained either by introgression following embryonic stem (ES) cell manipulations or direct use of nucleases. These apparently contradictory observations could result from a genetic compensation in invalidated animals [5] or from an increased robustness [6].

In the present report, we comparatively assessed, at the transcriptomic level, the impact of *Prnp* and *Sprn* knock-out in E6.5 mouse embryos and its consequences following inoculation with *ShRNA*-lentiviral vectors at the one cell stage.

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<footnote>*Correspondence: andrea.rau@inrae.fr</footnote>

<footnote n="1">¹ Université Paris-Saclay, INRAE, AgroParisTech, GABI, 78350 Jouy-en-Josas, France
Full list of author information is available at the end of the article</footnote>
Transgenic lines and lentiviral inoculations

Transgenic *Prnp* and *Sprn* FVB/N knockout mouse lines were already described [2, 4, 7]. Wild type (WT) FVB/N mice were purchased from Janvier [8]. ShRNA lentiviral vector solutions were purchased from Sigma with infectious titers over 10⁹ infectious units/mL (LS1: TRCN0000179960 and LS2: TRCN0000184740 against *Sprn* transcripts, LP1: TRCN0000319687 and LP2: TRCN 0000273801 against *Prnp* transcripts). Intra-perivitel- lin space injections and transplantation into pseudo- pregnant recipient mice were performed as previously described [2]. Around 50 one-cell stage embryos were injected for each genotype and lentiviral solution combi- nation (Figure 1A).

Embryo collection and transcriptomic analyses

Embryos were collected at E6.5 (Figure 1B). Total RNA was isolated from pools of 6–14 E6.5 embryos, deriving from 3 to 5 females. RNA extractions and integrity analysis were performed as previously described [2]. Three to four independent pools were produced for each experimental group (i.e., genotype and lentiviral solution combination) and analysed using Agilent SurePrint G3 gene expression V2 8 × 60 K mouse microarrays (AMADID: 074809, Figure 1C). All steps were performed by the @ BRIDGe facility (INRAE Jouy-en-Josas, France), as described previously [9].

All analyses were performed with R version 4.0.0.
Median pixel intensity and local background intensity
were read and pre-processed from the raw Agilent files
using the R/Bioconductor package *limma* (version 3.44.1,
[10, 11]). Probe intensities were quantile-normalized and
log₂-transformed [12]. Using the "gIsWellAboveBack-
ground" flag, non-control probes were called as present
if they were above background in at least 3 samples.
After averaging intensities for remaining probes with
identical target sequences, a single representative probe

was chosen for each gene according to the maximum
observed variance across samples (Figure 1C).

Hallmark gene set analyses

To evaluate the potential role played by specific ensem-
bles of gene sets, hallmark gene sets from the Molecular
Signatures Database (MSigDB, [13, 14]) were obtained
for *Mus musculus* using the *msigdbr* package (version
7.2.1). Among the 50 available gene sets, we focused
our attention on a subset of 14 hallmark gene sets
related to PrP recognized physiological functions (see
below). Comparisons of interest for the hallmark gene
set analysis were defined for four different experimental
groups as compared to WT mice: (1) *Prnp*<sup>0/0</sup>*Sprn*<sup>0/0</sup>; (2) *Prnp*<sup>0/0</sup>*Sprn*<sup>0/0</sup>*Sprn*<sup>KD</sup>; (3) *Prnp*<sup>0/0</sup>*Sprn*<sup>0/0</sup>*Prnp*<sup>KD</sup>; and (4) *Prnp*<sup>0/0</sup>*Sprn*<sup>KD</sup>. To minimize possible off-target effects,
contrasts for comparisons with groups (2)-(4) were con-
structed by averaging over the two lentiviruses for each
gene knockdown. Using the *fry* self-contained rotation
gene set test from *limma* [15], we sought to identify
whether genes in each selected hallmark gene set were
globally differentially expressed for a given comparison
(Figure 1D). *P*-values were calculated corresponding to
tests for gene sets exhibiting significant over-expression
(“Up”) and under-expression (“Down”), as well as dif-
ferential expression regardless of direction (“Mixed”).
Raw *P*-values were corrected for multiple testing using
the Benjamini–Hochberg approach to control the false
discovery rate (FDR, [16]), and gene sets were identified
as significantly globally differentially expressed if their
adjusted *P*-value < 0.05.

Differential expression analysis

For the differential analysis (Figure 1E), a linear model
with group-means parameterization (i.e., no intercept
and a separate coefficient for each group) was fit for each
gene. Using *limma*, an empirical Bayes approach was
used to moderate the standard errors of the estimated

<figure><img src="image_6.png" /><figcaption>**Figure 1** Schematic representation of study design and analysis. **A** Transgenic lines and lentiviral inoculations. **B** Embryo collection. **C** Transcriptomic analyses. **D** Hallmark gene set analysis; and **E** differential expression analysis.</figcaption></figure>
log-fold changes. Contrasts were defined to identify differentially expressed genes for each comparison of interest; we focused in particular on the comparison of *Prnp*<sup>0/0</sup>*Sprn*<sup>0/0</sup> and WT E6.5 embryos. As before, *P*-values were corrected for multiple testing using the Benjamini-Hochberg approach to control the false discovery rate [16], and genes were identified as significantly differentially expressed if their adjusted *P*-value < 0.05 and absolute log fold change > 1.

### RT-qPCR analysis

Reverse transcription was performed on 500 ng of total RNA from the 4 pools of WT and the 3 pools of *Prnp*<sup>0/0</sup>*Sprn*<sup>0/0</sup> E6.5 embryos previously used for transcriptomic analysis (see the “Embryo collection and transcriptomic analyses” section) using InVitrogen SuperScript™ IV Vilo™ reverse transcriptase kit (11766500) and random primers, according to the manufacturer’s instructions. RT-qPCR quantification was performed on triplicates using the SYBR Green quantitative PCR kit (Applied Biosystems) and standard PCR conditions. Primers were designed on separate exons to produce 100-bp amplicons, with a Tm of 60 °C. Analyses were performed using the Δ(ΔCt) method (Biogazelle QBasePlus software, Biogazelle NV, Ghent, Belgium) and normalizing genes. The GAPDH and UBC genes were used for normalization, using primers 5'-tgacgtgc-cgcctggagaaa-3' and 5'-agtgtagccaagatgcccttcag-3' for GAPDH and 5'-cgtcgagcccagtgttaccaccaagaagg-3' and 5'-cccccatcacacccaagaacaagcacaag-3' for UBC. Three

genes were included for RT-qPCR analyses: *Ada*, using primers 5'-tagacactgactaccagatgac-3' and 5'-tggctattggtattctctgtag-3'; *Cds2*, using primers 5'-tggatcgctttgactgccagt-3' and 5'-tgttgaagatgtgaagctgctg-3'; and *Spint1*, using primers 5'-aggaacagcagtgcttgtag-3' and 5'-atgcagatgcaacgaaatacag-3'.

### Analysis results

Although *Prnp*<sup>0/0</sup>*Sprn*<sup>0/0</sup> mice are viable [3, 4], the knockdown of *Sprn* in *Prnp*<sup>0/0</sup> mouse embryos was reported to induce embryonic lethality highlighted by a developmental failure of the trophectoderm-derived compartment noticeable at E7.5 [2]. We reinvestigated this latter observation by transcriptomic analysis of such embryos at E6.5, focusing on a subset of MSigDB including 14 hallmark gene sets related to PrP recognized physiological functions ([1, 17–19], Table 1). Three of those hallmark gene sets were significantly altered in *Sprn*-knockdown, *Prnp*<sup>0/0</sup> E6.5 embryos compared to their WT counterparts (adjusted *P*-value < 0.05): interferon-α and -γ responses and apoptosis, while inflammatory response was significant with an adjusted *P*-value < 0.10 (Table 1).

We similarly investigated the transcriptomic outcomes at E6.5 of *Sprn*- or *Prnp*-knockdown in *Prnp*<sup>0/0</sup>*Sprn*<sup>0/0</sup> mouse embryos. The knockdowns of *Sprn* or *Prnp* were performed on a knockout genotype for both of these genes to highlight only those pathways associated with lentiviral ShRNA vector infections on this specific genetic background. Two different ShRNAs were again used for each targeted gene to reduce the likelihood of observing

<table><caption>**Table 1 Hallmark gene set analyses at E6.5.**</caption><thead><tr><th rowspan="2">Hallmark gene set</th><th colspan="2">WT vs P0S0</th><th colspan="2">WT vs P0S0S-</th><th colspan="2">WT vs P0S0P-</th><th colspan="2">WT vs P0S-</th></tr><tr><th>Direction</th><th>FDR</th><th>Direction</th><th>FDR</th><th>Direction</th><th>FDR</th><th>Direction</th><th>FDR</th></tr></thead><tbody><tr><td>Adipogenesis</td><td>Up</td><td>0.62552876</td><td>Up</td><td>0.80753343</td><td>Down</td><td>0.79673023</td><td>Up</td><td>0.79147888</td></tr><tr><td><strong>Apoptosis</strong></td><td>Down</td><td>0.93132096</td><td>Down</td><td>0.17156136</td><td>Down</td><td>0.16934382</td><td><strong>Down</strong></td><td><strong>0.01640416</strong></td></tr><tr><td>Cholesterol homeostasis</td><td>Up</td><td>0.90561461</td><td>Down</td><td>0.85373899</td><td>Down</td><td>0.49413621</td><td>Down</td><td>0.71885689</td></tr><tr><td>E2F targets</td><td>Down</td><td>0.90561461</td><td>Up</td><td>0.19319905</td><td>Up</td><td>0.31979891</td><td>Up</td><td>0.11180027</td></tr><tr><td>Epithelial mesenchymal transition</td><td>Down</td><td>0.93132096</td><td>Down</td><td>0.4305476</td><td>Down</td><td>0.31979891</td><td>Down</td><td>0.1308924</td></tr><tr><td>Hypoxia</td><td>Up</td><td>0.90561461</td><td>Down</td><td>0.42575279</td><td>Down</td><td>0.18867174</td><td>Down</td><td>0.11180027</td></tr><tr><td>Inflammatory response</td><td>Up</td><td>0.90561461</td><td>Down</td><td>0.10111071</td><td>Down</td><td>0.18867174</td><td>Down</td><td>0.07054624</td></tr><tr><td><strong>Interferon alpha response</strong></td><td>Down</td><td>0.90561461</td><td><strong>Down</strong></td><td><strong>0.00193216</strong></td><td><strong>Down</strong></td><td><strong>0.00379274</strong></td><td><strong>Down</strong></td><td><strong>0.00041329</strong></td></tr><tr><td><strong>Interferon gamma response</strong></td><td>Down</td><td>0.90561461</td><td><strong>Down</strong></td><td><strong>0.00463537</strong></td><td><strong>Down</strong></td><td><strong>0.00972725</strong></td><td><strong>Down</strong></td><td><strong>0.00075138</strong></td></tr><tr><td>Notch signaling</td><td>Down</td><td>0.90561461</td><td>Up</td><td>0.80753343</td><td>Up</td><td>0.82613941</td><td>Down</td><td>0.9957742</td></tr><tr><td><strong>Reactive oxygen species pathway</strong></td><td>Up</td><td>0.90561461</td><td><strong>Down</strong></td><td><strong>0.01564074</strong></td><td>Down</td><td>0.18867174</td><td>Down</td><td>0.17771965</td></tr><tr><td>TGF beta signaling</td><td>Up</td><td>0.90561461</td><td>Down</td><td>0.57494044</td><td>Down</td><td>0.557864</td><td>Down</td><td>0.79147888</td></tr><tr><td>Wnt beta catenin signaling</td><td>Down</td><td>0.90561461</td><td>Up</td><td>0.19319905</td><td>Up</td><td>0.18867174</td><td>Up</td><td>0.10479931</td></tr><tr><td>Xenobiotic metabolism</td><td>Up</td><td>0.62552876</td><td>Down</td><td>0.80753343</td><td>Down</td><td>0.68538223</td><td>Up</td><td>0.95587952</td></tr></tbody></table>

Top margin: Compared genotypes. P0: *Prnp*<sup>0/0</sup>, SO: *Sprn*<sup>0/0</sup>, S-: knockdown of *Sprn*. P-: knockdown of *Prnp*. For each knockdown, two independent lentiviral ShRNA vectors were used (see the “Transgenic lines and lentiviral inoculations” section). Left margin: hallmark gene sets [13, 14]. Significantly altered hallmark gene sets are highlighted in boldface (FDR < 0.05) and italicized (FDR < 0.10)
an off-target-induced biological disturbance. Compared to WT E6.5 embryos, only two hallmark gene sets were consistently and significantly altered: interferon-α and -γ responses (Table 1). However, compared to the previous analysis, the statistical significance of these gene sets was unexpectedly reduced by tenfold. Furthermore, no apoptosis induction was detected (Table 1).

We next compared the transcriptome of E6.5 WT and *Prnp*<sup>0/0</sup>*Sprn*<sup>0/0</sup> mouse embryos. Only 11 genes were found to be differentially expressed between these two genotypes with an adjusted *P*-value < 0.05 and absolute log fold change > 1 (Table 2). All 11 of these genes were similarly found to be significantly differentially expressed in the same direction in *Sprn*-knockdown, *Prnp*<sup>0/0</sup> compared to WT E6.5 mouse embryos, albeit with weaker log fold changes for the majority. As already discussed, the *Prnp* and *Sprn* gene invalidations did not induce alteration of their transcript expression levels, and their absence in this list was thus expected [4, 7]. Most of the differentially expressed genes were reported to be transcribed in the embryo ectoderm and mesenchyme, and only a few in the endoderm or in the extraembryonic component (Table 2, [20]).

Finally, we focused our attention on the three genes expressed in the extraembryonic component (*Spint1*, *Cds2*, *Ada*); in particular, *Ada* exhibited strong differential expression (log fold-change = -2.1, Table 2, Figure 2). Differential expression of these three genes was further assessed by RT-qPCR analysis. The results validated those obtained from the microarray data for *Ada* and *Cds2*; a similar but insignificant trend was observed for *Spint1* (Table 2).

## Discussion

Our results confirmed that knockdowns of *Sprn* in *Prnp*<sup>0/0</sup> mouse embryos induce apoptosis alongside interferon responses at E6.5, in accord with previous reports and suggesting that embryonic lethality could be diagnosed at earlier developmental stages. Because two different ShRNAs were used, targeting different regions of the *Sprn* transcript, it is unlikely that apoptosis results from an off-target effect.

A natural subsequent question is whether this apoptotic induction, alongside inflammatory and interferon responses, could result from the association of a lentiviral ShRNA-expressing vector inoculation [21] with a *Prnp*-knockout induced interferon-primed state [22] in the absence/reduction of *Sprn* expression, which has been shown to be required to induce apoptosis [2]. In our study, we found that *Prnp* or *Sprn* knockdown in mouse *Prnp*<sup>0/0</sup>*Sprn*<sup>0/0</sup> embryos induces reduced interferon responses and no apoptosis at E6.5. These results could suggest that the expression or the knockout of *Sprn*

is required to avoid lentiviral ShRNA vector induction of a strong interferon response associated with apoptosis in *Prnp*<sup>0/0</sup> mouse embryos, while its knockdown exacerbates these pathways. A potential explanation for these apparent contradictory observations is that the knock-out of the two genes induces a genetic adaptation that in turn helps control the lentiviral-induced responses. Such an adaptation might not take place with the *Sprn*-knockdown or to an insufficient level.

To assess this hypothesis, we compared the gene expression of WT and *Prnp*<sup>0/0</sup>*Sprn*<sup>0/0</sup> E6.5 embryos, revealing highly similar transcriptomes with only 11 differentially expressed genes. Since adult expression of both *Prnp* and *Sprn* genes is more abundant in the nervous system, and since PrP involvement in muscle and bone development/regeneration has been previously reported, deregulation of these genes in the ectoderm and in the mesenchyme might be relevant observations. However, in *Sprn*-knockdown, *Prnp*<sup>0/0</sup> embryos, a developmental failure of the trophectoderm-derived compartment was reported [2], instead suggesting a major role of the extraembryonic component in the appearance of this lethality. Only 3 out of the 11 differentially expressed genes (*Spint1*, *Cds2*, *Ada*) are known to be expressed in the extraembryonic component. *Spint1* was recently reported to be a biomarker of placental insufficiency [23]. Low circulating levels of *Spint1* are associated with placental failure whereas here, at E6.5, this expression is higher in *Prnp*<sup>0/0</sup>*Sprn*<sup>0/0</sup> embryos compared to their WT counterparts. Whether *Spint1* overexpression can favor placental development remains to be demonstrated. *Cds2* is a widely expressed gene indirectly involved in the positive control of angiogenesis [24]. Its overexpression in *Prnp*<sup>0/0</sup>*Sprn*<sup>0/0</sup> embryos could suggest a sustained angiogenesis of the placenta, but in the absence of associated deregulation of co-factors, such as vascular endothelial growth factors, the interpretation of this observation remains fragile. Nevertheless, the differential expression of the two above-mentioned genes appears to favor placental development and to contribute to the survival of the *Prnp*<sup>0/0</sup>*Sprn*<sup>0/0</sup> mouse embryos. However, their potential implication in the control of the interferon response remains elusive.

The third strongly differentially expressed gene transcribed in the extraembryonic component was *Ada*. Disruption of the *Ada* gene in mice induces perinatal lethality [25], a phenotype rescued by tissue-specific placental expression of this gene [26]. Its crucial role in the trophectoderm-derived compartment was also indirectly emphasized through the knockout of the AP-2γ transcription factor-encoding gene that resulted in an early embryonic lethal phenotype, similar to that observed for *Sprn*-knockdown in *Prnp*<sup>0/0</sup> embryos [2, 27], associated
<table><caption>**Table 2** Differentially expressed genes between *Prnp*<sup>0/0</sup>*Sprn*<sup>0/0</sup> and WT E6.5 mouse embryos.</caption><thead><tr><th>Gene name</th><th>Description</th><th>NCBI gene</th><th>Log fold change</th><th>Adjusted P-value</th><th>Embryo ectoderm</th><th>Embryo endoderm</th><th>Embryo mesenchyme</th><th>Extraembryonic component</th><th>RT-qPCR Prnp<sup>0/0</sup> Sprn<sup>0/0</sup> E6.5</th><th>RT-qPCR WT E6.5</th><th>RT-qPCR P-value</th></tr></thead><tbody><tr><td>Spint1</td><td>Serine protease inhibitor, Kunitz type 1</td><td>20732</td><td>1.599051</td><td>1.96414E-08</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td><td>29.56±2.1</td><td>25.88±4.5</td><td>0.126</td></tr><tr><td>Gm30906</td><td>Long non-coding RNA</td><td>102632964</td><td>-1.477668</td><td>1.96414E-08</td><td>✓</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Scg5</td><td>Secretogranin V, secreted chaperone protein</td><td>20394</td><td>-1.334427</td><td>4.2595E-08</td><td>✓</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Spg11</td><td>Spatacsin vesicle trafficking associated</td><td>214585</td><td>-1.081024</td><td>2.05026E-06</td><td>✓</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Cds2</td><td>CDP-diacetylglycerol synthase 2</td><td>110911</td><td>1.297156</td><td>2.42507E-06</td><td>✓</td><td></td><td></td><td>✓</td><td>50.87±5.5</td><td>26.31±4.8</td><td>0.001</td></tr><tr><td>Jmjd7</td><td>Jumonji domain containing 7</td><td>433466</td><td>-1.479236</td><td>1.31005E-05</td><td>✓</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Ada</td><td>Adenosine deaminase</td><td>11486</td><td>-2.117557</td><td>4.32324E-05</td><td>✓</td><td></td><td></td><td>✓</td><td>10.53±1.1</td><td>16.08±4.3</td><td>0.043</td></tr><tr><td>AK148702</td><td>RIKEN clone 7120437D13 (MGI:3537747)</td><td></td><td>2.819679</td><td>0.004937838</td><td>-</td><td>-</td><td>-</td><td>-</td><td></td><td></td><td></td></tr><tr><td>Cplx2</td><td>Complexin 2</td><td>12890</td><td>2.616736</td><td>0.010457547</td><td>✓</td><td></td><td></td><td>✓</td><td></td><td></td><td></td></tr><tr><td>Gm10734</td><td>RIKEN clone I530011G18 (MGI:3565867)</td><td></td><td>-1.104109</td><td>0.010457547</td><td>✓</td><td></td><td></td><td>✓</td><td></td><td></td><td></td></tr><tr><td>Sdc4</td><td>Syndecan 4</td><td>20971</td><td>-1.157117</td><td>0.021156447</td><td>✓</td><td></td><td></td><td>✓</td><td></td><td></td><td></td></tr></tbody></table>

Results are shown for significantly differentially expressed genes (FDR <0.05, absolute log fold change ≥ 1). Checkmarks for each gene represent reported expression in embryo ectoderm, embryo endoderm, embryo mesenchyme, and extraembryonic component [20]. Blank spaces and dashes represent unreported expression and no available data, respectively. Normalized RT-qPCR expression levels are reported in each group for the three tested genes (mean ± standard errors) with the associated P-value from a two-sample Student's t-test.
<figure><img src="image_7.png" /><figcaption>**Figure 2** Normalized expression of *Ada* in WT; *Prnp*<sup>0/0</sup>*Sprn*<sup>0/0</sup>; and *Prnp*<sup>0/0</sup>*Sprn*<sup>KD</sup> mice. Values are represented as dot plots for individual samples (grey points) with means (black points) and standard deviations (bars) for each experimental group.</figcaption></figure>

with a lack of *Ada* gene expression in the extraembryonic cells [28]. However, in *Prnp*<sup>0/0</sup>*Sprn*<sup>0/0</sup> mouse embryos, only a downregulation of the *Ada* gene expression is observed, thus likely avoiding the occurrence of these drastic phenotypes. It should be mentioned that *Ada*<sup>0/+</sup> mouse embryos were similarly not reported to be affected [25]. Interestingly, Ada congenital defect induces a severe combined immunodeficiency syndrome [27]. Expression levels of this enzyme correlate with the production levels of interferons and proinflammatory factors, and modulation of Ada activity was even proposed as a potential therapeutic target [28–31]. High interferon responses can induce side effects among which some, such as autoimmune reactions, can be detrimental. The control of the interferon response is thus crucial, and as already mentioned, altered in the absence of members of the prion protein family [17]. The downregulation of the *Ada* gene expression observed in *Prnp*<sup>0/0</sup>*Sprn*<sup>0/0</sup> mouse embryos might help to control the interferon and inflammatory responses induced by lentiviral ShRNA-encoding vector infections to a level compatible with their survival. This genetic adaptation is only partially induced in *Sprn* knockdown, *Prnp*<sup>0/0</sup> embryos, resulting in a high rate of embryonic lethality [2].

An alternative explanation would be that expression of a ShRNA targeting the invalidated gene in knockout mice does not induce specific immune response and antiviral functions due to the absence of knockdown-induced RNA degradation products [32]. However, the design of the *Prnp* and *Sprn* gene invalidations was such that their transcription remains unaffected, while the produced mRNA, which still encompasses the ShRNA target site, no longer encodes for the protein. As RNA expression was confirmed at embryonic stages for *Prnp* [33] and *Sprn* [7] knockout mice, this hypothesis is thus unlikely.

Overall, our results suggest a genetic adaptation of *Prnp*<sup>0/0</sup>*Sprn*<sup>0/0</sup> mouse embryos, both to sustain placental physiology that is affected in the absence of PrP [31] or Shadoo [7] and to refrain the upregulation of induced interferon responses following environmental stresses. This genetic adaptation might involve the downregulation of *Ada* and its related pathways, this protein being involved in immunomodulation and ectoplacental development. Although this hypothesis remains to be further supported by direct experiments, it offers an explanation for the discrepancy observed between knockdowns and knockouts in previously reported data [2, 3] and adds to the list of knockout genotypes that have acquired genetic adaptation.

**Authors' contributions**

AR, FJ, KMG, and JLV conceived the study. BP, JC, JL, and KMG collected mouse embryos and performed RNA extractions. JL performed all laboratory steps related to microarray analyses and NDC performed the RT-qPCR experiments. AR, AA, MM, FJ, and DL performed bioinformatic and statistical analyses of the transcriptomics data. AR, BP, KMG, and JLV drafted the manuscript and prepared tables and figures. All authors read and approved the final manuscript.

**Funding**

This work was supported by an INRAE Animal Genetics department project grant (grant AAP.GA.2015, COSI-Net).

**Availability of data and materials**

Raw microarray data files and all analysis scripts needed to pre-process the data and reproduce the analyses described in this work are openly available on the Data INRAE portal [34].

**Declarations**

**Ethics approval and consent to participate**

Animal experiments were carried out in accordance with the EU Directive 2010/63/EU. Production, breeding and inoculations at the one cell stage with *ShRNA*-lentiviral vectors of the various transgenic lines were approved by the Ethics Committee of Jouy-en-Josas (Comethea, Permit number 02532.01), and followed the safety recommendations of the French "Haut Conseil des Biotechnologies" (HCB, Permit numbers 6460 and 5468).

**Competing interests**

The authors declare that they have no competing interests.

**Author details**

¹Université Paris-Saclay, INRAE, AgroParisTech, GABI, 78350 Jouy-en-Josas, France. ²BioEcoAgro Joint Research Unit, INRAE, Université de Liège, Université de Lille, Université de Picardie Jules Verne, 80203 Péronne, France.

Received: 6 May 2022 Accepted: 13 June 2022

Published online: 07 July 2022

**References**

1. Onodera T, Nishimura T, Sugiura K, Sakudo A (2020) Function of prion protein and the family member, Shadoo. Curr Issues Mol Biol 36:67–88. https://doi.org/10.21775/cimb.036.067

2. Passet B, Young R, Makhzami S, Vilotte M, Jaffrézic F, Halliez S, Bouet S, Marthey S, Khalifé M, Kanelopoulos-Langevin C, Béringue V, Le Provost F, Laude H, Vilotte JL (2012) Prion protein and Shadoo are involved in overlapping embryonic pathways and trophoblastic development. PLoS One 7:e41959. https://doi.org/10.1371/journal.pone.0041959
3. Daude N, Wohlgemuth S, Brown R, Pitstick R, Gapeshina H, Yang J, Carlson GA, Westaway D (2012) Knockout of the prion protein (PrP)-like *Sprn* gene does not produce embryonic lethality in combination with PrP<sup>C</sup>-deficiency. Proc Natl Acad Sci USA 109:9035–9040. https://doi.org/10.1073/pnas.1202130109

4. Castille J, Passet B, Makhzami S, Vilotte M, Moazami-Goudarzi K, Truchet S, Daniel-Carlier N, Gaillard AL, Andréoletti O, Vaiman D, Beauvallet C, Vaiman A, Floriot S, Calvel P, Mouillet-Richard S, Duchesne A, Béringue V, Vilotte JC (2021) Co-invalidation of *Pnrp* and *Sprn* in FVB/N mice affects reproductive performances and highlight complex biological relationship between PrP and Shadoo. Biochem Biophys Res Commun 551:1–6. https://doi.org/10.1016/j.bbkrc.2021.03.013

5. Rossi A, Kontarakis Z, Gerri C, Nolte H, Hölper S, Krüger M, Stainier DYR (2015) Genetic compensation induced by deleterious mutations but not gene knockdowns. Nature 524:230–233. https://doi.org/10.1038/natur e14580

6. Schlieper G, Kim JH, Molojavyi A, Jacoby C, Laussmann T, Flögel U, Gödecke A, Schrader J (2004) Adaptation of the myoglobin knockout mouse to hypoxic stress. Am J Physiol Regul Integr Comp Physiol 286:R786-792. https://doi.org/10.1152/ajpregu.00043.2003

7. Passet B, Castille J, Makhzami S, Truchet S, Vaiman A, Floriot S, Moazami-Goudarzi K, Vilotte M, Gaillard AL, Helary L, Bertaud M, Andréoletti O, Vaiman D, Calvel P, Daniel-Carlier N, Moudjou M, Beauvallet C, Benharouga M, Laloë D, Mouillet-Richard S, Duchesne A, Béringue V, Vilotte JL (2020) The Prion-like protein Shadoo is involved in mouse embryonic and mammary development and differentiation. Sci Rep 10:6765. https://doi.org/10.1038/s41598-020-63805-y

8. Janvier Labs FVB mouse model. https://janvier-labs.com/en/ficheProdu it/fvb一体 mouse. Accessed 22 June 2022

9. Mach N, Ramayo-Caldas Y, Clark A, Moroldo M, Robert C, Barrey E, López JM, Le Moyec L (2017) Understanding the response to endurance exercise using a systems biology approach: combining blood metabolomics, transcriptomics and miRNomics in horses. BMC Genomics 18:187. https:// doi.org/10.1186/s12864-017-3571-3

10. Ritchie ME, Phipson B, Wu D, Hu Y, Law CW, Shi W, Smyth GK (2015) *limma* powers differential expression analyses for RNA-sequencing and microarray studies. Nucleic Acids Res 43:e47. https://doi.org/10.1093/nar/gkv007

11. Phipson B, Lee S, Majewski LJ, Alexander WS, Smyth GK (2016) Robust hyperparameter estimation protects against hypervariable genes and improves power to detect differential expression. Ann Appl Stat 10:946–963. https://doi.org/10.1214/16-AOAS920

12. Ritchie ME, Silver J, Oshlack A, Holmes M, Diyagama D, Holloway A, Smyth GK (2007) A comparison of background correction methods for two-colour microarrays. Bioinformatics 23:2700–2707. https://doi.org/10.1093/bioinformatics/btm412

13. Subramanian A, Tamayo P, Mootha VK, Mukherjee S, Ebert BL, Gillette MA, Paulovich A, Pomeroy SL, Golub TR, Lander ES, Mesirov JP (2005) Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc Natl Acad Sci USA 102:15545– 15550. https://doi.org/10.1073/pnas.0506580102

14. Liberzon A, Birger C, Thorvaldsdóttir H, Ghandi M, Mesirov JP, Tamayo P (2015) The Molecular Signatures Database (MSigDB) hallmark gene set collection. Cell Syst 1:417–425. https://doi.org/10.1016/j.cels.2015.12.004

15. Wu D, Lim E, Vaillant F, Asselin-Labat ML, Visvader JE, Smyth GK (2010) ROAST: rotation gene set tests for complex microarray experiments. Bioinformatics 26:2176–2182. https://doi.org/10.1093/bioinformatics/btq401

16. Benjamini Y, Hochberg J (1995) Controlling the false discovery rate: a practical and powerful approach to multiple testing. J R Stat Soc B 57:289–300. https://doi.org/10.1111/j.2517-6161.1995.tb02031.x

17. Salvesen Ø, Tatzelt J, Tranulis MA (2019) The prion protein in neuroim- mune crosstalk. Neurochem Int 130:104335. https://doi.org/10.1016/j. neuint.2018.11.010

18. Sakaguchi S, Chida J (2020) Prion protein is a novel modulator of influ- enza: potential implications for anti-influenza therapeutics. Curr Issues Mol Biol 37:21–32. https://doi.org/10.21775/cimb.037.021

19. Schmitt-Ulms G, Mehrabian M, Williams D, Ehsani S (2021) The IDIP framework for assessing protein function and its application to the prion protein. Biol Rev Camb Philos Soc 96:1907–1932. https://doi.org/10.1111/ brv.12731

20. Mouse Genome Informatics (MGI) Gene eXpression Database (GXD). http://www.informatics.jax.org/expression.shtml. Accessed 22 June 2022

21. Aldo PB, Mulla MJ, Romero R, Mor G, Abrahams VM (2010) Viral ssRNA induces first trimester trophoblast apoptosis through an inflammatory mechanism. Am J Reprod Immunol 64:27–37. https://doi.org/10.1111/j. 1600-0897.2010.00817.x

22. Malachin G, Reiten MR, Salvesen Ø, Aanes H, Kamstra JH, Skovgaard K, Heegaard PMH, Ersdal C, Espenes A, Tranulis MA, Bakkebø MK (2017) Loss of prion protein induces a primed state of type I interferon-responsive genes. PLoS One 12:e0179881. https://doi.org/10.1371/journal.pone. 0179881

23. Kaitu'u-Lino TJ, MacDonald TM, Cannon P, Nguyen TV, Hiscock RJ, Haan N, Myers JE, Hastie R, Dane KM, Middleton AL, Bittar I, Sferruzzi-Perri AN, Pritchard N, Harper A, Hannan NJ, Kyritsis V, Crinis N, Hui L, Walker SP, Tong S (2020) Circulating SPINT1 is a biomarker of pregnancies with poor placental function and fetal growth restriction. Nat Commun 11:2411. https://doi.org/10.1038/s41467-020-16346-x

24. Zhao W, Cao L, Ying H, Zhang W, Li D, Zhu X, Xue W, Wu S, Cao M, Fu C, Qi H, Hao Y, Tang YC, Qin J, Zhong TP, Lin X, Yu L, Li X, Li L, Wu D, Pan W (2019) Endothelial CDS2 deficiency causes VEGFA-mediated vascular regression and tumor inhibition. Cell Res 29:895–910. https://doi.org/10.1038/ s41422-019-0229-5

25. Wakamiya M, Blackburn MR, Jurecic R, McArthur MJ, Geske RS, Cartwright JJ, Mitani K, Vaishnav S, Belmont JW, Kellems RE (1995) Disruption of the adenosine deaminase gene causes hepatocellular impairment and perinatal lethality in mice. Proc Natl Acad Sci USA 92:3673–3677. https:// doi.org/10.1073/pnas.92.9.3673

26. Blackburn MR, Wakamiya M, Caskey CT, Kellems RE (1995) Tissue-specific rescue suggests that placental adenosine deaminase is important for fetal development in mice. J Biol Chem 270:23891–23894. https://doi. org/10.1074/jbc.270.41.23891

27. Werling U, Schorle H (2002) Transcription factor gene AP-2 gamma essen- tial for early murine development. Mol Cell Biol 22:3149–3156. https://doi. org/10.1128/MCB.22.9.3149-3156.2002

28. Pérez-Aguilar MC, Goncalves L, Ibarra A, Bonfante-Cabarcas R (2010) Adenosine deaminase as costimulatory molecule and marker of cellular immunity. Invest Clin 51:561–571

29. Yoneyama Y, Sawa R, Suzuki S, Miura A, Kobayashi H, Doi D, Yoneyama K, Araki T (2002) Relation between adenosine deaminase activities and cytokine-producing T cells in women with preeclampsia. Clin Biochem 35:303–306. https://doi.org/10.1016/s0009-9120(02)00318-1

30. Tang R, Ma CS, Dong JZ, Liu XP, Liu X (2006) Does adenosine deaminase play a key role in coronary artery disease. Med Hypotheses 67:371–374. https://doi.org/10.1016/j.mehy.2006.01.038

31. Kutryb-Zajac B, Mierzejewska P, Slominska EM, Smolenski RT (2020) Thera- peutic perspectives of adenosine deaminase inhibition in cardiovascular diseases. Molecules 25:4652. https://doi.org/10.3390/molecules25204652

32. Meng Z, Qiu S, Zhang X, Wu J, Schreiter T, Xu Y, Yang D, Roggendorf M, Schlaak J, Lu M (2009) Inhibition of woodchuck hepatitis virus gene expression in primary hepatocytes by siRNA enhances the cellular gene expression. Virology 384:88–96. https://doi.org/10.1016/j.virol.2008.11.012

33. Khalifé M, Young R, Passet B, Halliez S, Vilotte M, Jaffrézic F, Marthey S, Béringue V, Vaiman D, Le Provost F, Laude H, Vilotte JL (2011) Transcrip- tomic analysis brings new insight into the biological role of the prion protein during mouse embryogenesis. PLoS One 6:e23253. https://doi. org/10.1371/journal.pone.0023253

34. Rau A, Passet B, Castille J, Daniel-Carlier N, Asset A, Lecardonnel J, Mor- oldo M, Jaffrézic F, Laloe D, Moazami-Goudarzi K, Vilotte JL (2022) Sup- plementary data: potential genetic robustness of *Pnrp* and *Sprn* double knockout mouse embryos towards ShRNA-lentiviral inoculation. Data INRAE portal. https://doi.org/10.15454/1L50VK

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