Human Somatostatin SST4 Receptor Transgenic Mice: Construction and Brain Expression Pattern Characterization

Somatostatin receptor subtype 4 (SST4) has been shown to mediate analgesic, antidepressant and anti-inflammatory functions without endocrine actions; therefore, it is proposed to be a novel target for drug development. To overcome the species differences of SST4 receptor expression and function between humans and mice, we generated an SST4 humanized mouse line to serve as a translational animal model for preclinical research. A transposon vector containing the hSSTR4 and reporter gene construct driven by the hSSTR4 regulatory elements were created. The vector was randomly inserted in Sstr4-deficient mice. hSSTR4 expression was detected by bioluminescent in vivo imaging of the luciferase reporter predominantly in the brain. RT-qPCR confirmed the expression of the human gene in the brain and various peripheral tissues consistent with the in vivo imaging. RNAscope in situ hybridization revealed the presence of hSSTR4 transcripts in glutamatergic excitatory neurons in the CA1 and CA2 regions of the hippocampus; in the GABAergic interneurons in the granular layer of the olfactory bulb and in both types of neurons in the primary somatosensory cortex, piriform cortex, prelimbic cortex and amygdala. This novel SST4 humanized mouse line might enable us to investigate the differences of human and mouse SST4 receptor expression and function and assess the effects of SST4 receptor agonist drug candidates.

Previous studies used Sstr4 knockout (KO) and wild-type (WT) mice, since no SST4 receptor specific antagonist is commercially available. Investigating Sstr4 KO mouse models and the synthetic SST 4 receptor agonist J-2156, we and other workgroups provided evidence that the SST 4 receptor is a unique novel drug target for the treatment of chronic pain and depression [2,4,20,[24][25][26][27].
The presently available drugs for these conditions are often not efficacious enough and cause serious side effects upon long-term use [28]. Thus, the SST 4 receptor has recently come into the focus of interest of drug development, and pharmaceutical companies have started to develop non-peptide SST 4 agonists [29]. The agonist design has been greatly enhanced by the in silico 3D modeling of the human receptor structure [30,31].
Humanized mice are predominantly generated by engrafting human cells, tissues and tumors and used extensively for immunological and oncological research [32][33][34][35][36][37], but genetic modification is also an increasingly applied method [38][39][40][41]. Human receptor expressing mouse models are useful in translational drug research, providing more predictive results relevant for the human diseases and more relevant platforms for testing drug candidates [42,43]. For example, Fox et al. replaced the native bradykinin B 1 receptor gene in mouse with its human B 1 receptor gene to investigate the human B 1 receptorspecific antagonist NVP-SAA164 in vivo after the successful in vitro results. NVP-SAA164 showed an anti-hyperalgesic function in these humanized mice, but not in WT nor in KO mice [44]. Jackson et al. found functional differences between mouse and human melanocortin receptor using MC1R humanized mice, such as the high ligand dependent eumelanogenesis in humanized mice. Mouse Mc1r receptor in WT mice in vivo, and both mouse and human melanocortin receptors in transfected cell lines in vitro showed ligand independent signaling [45].
The mouse SST 4 expression and function is relatively well-characterized in the brain [46], but little is known about the human receptor.
Therefore, our aims were to produce humanized mice by using a transposon vector containing the hSSTR4 gene with its regulatory elements, locate the random insertion sites and characterize the distribution pattern and the neuron populations expressing the transgene in the brain, as well as select the most useful mouse line for further functional research.

Vector Construction and Transgenesis
The final construct pPBC2 plasmid contained the full-length hSSTR4 gene with a 4-kb upstream and 2.7-kb downstream region of the human chromosome 20 to include every known regulatory element ( Figure 1A). At the end of the hSSTR4 coding sequence, luciferase and the tandem dimer Tomato (tdTomato) coding sequence were inserted, separated from the receptor by the P2A self-cleaving site, but the reporters were expected to be translated as a fusion protein. At the end of the downstream chromosomal region, a polyadenylation signal (polyA) sequence was added for transcription termination. The entire transgene cassette was flanked at both ends by Lox2272 Cre recombinase recognition sites as an option for conditional KO by insulators to inhibit position effects and by Piggy-Bac (PB) transposon inverted terminal repeats (ITR) as specific recognition sites for the PB transposase enzyme ( Figure 1B).
in late pregnancy. The third female produced 8 offspring (F1 generation), but it also died during its second pregnancy ( Figure 2D). In this founder mouse, the PB transposon integration sites were located by ligation-mediated polymerase chain reaction (LM-PCR) ( Figure 2E). From the F1 generation onwards, all mouse lines carried a single copy of the transgene, and none of those lines manifest a harmful phenotype based on viability, fertility and behavior. There were no differences between the litter sizes of the mouse lines (WT: 6.2 ± 0.4, KO: 5.8 ± 0.3, Chr3: 6.5 ± 0.4, U1: 6.0 ± 0.4, and U2: 5.9 ± 0.4). showing the full-length hSSTR4 coding sequence with up-and downstream regulatory elements (7.8 kb) as copied fragments and the neighboring genes (FOXA2 and THBD) (A). PB transposon vector carrying the upstream regulatory elements (4 kb) and human SSTR4 coding sequence followed by the P2A self-cleaving peptide sequence, luciferase and tdTomato coding sequences downstream SSTR4 regulatory elements (2.7 kb) and polyadenylation signal sequence (poly A). The entire transgenic cassette (12.7 kb) was flanked by the PB transposon inverted terminal repeats (ITR), insulators and Lox2272 Cre recombinase recognition sites (B).

Integration Sites of the hSSTR4 Transgene
The LM-PCR detected multiple insertion loci of the transgene in the F0 founder female. By sequencing of the LM-PCR products, three distinct integration sites were identified in chromosomes 3, 10 and X (copies were named Chr3, Chr10 and ChrX, respectively). The transgene inserted in the chromosome 10 did not pass onto the F1 generation ( Figure 2). The transgene in the chromosome 3 was inserted between the Sis and Otol1 genes, coding on the negative strand of the chromosome ( Figure 3). The PB transposase inserted the transposon while duplicating the TTAA recognition site, which started in the original position of 70,039,120 [47] (Figure 3). The transgene inserted in the chromosome 10 in the position of 67,335,940 [48], coding on the positive strand. The transgene inserted in the chromosome X at the position of 32,102,395 [49], coding on the positive strand. There were two mouse lines in which we were unable to locate the insertion site of the transgene (named U1 and U2) ( Figure 2).
To keep track of the transgene in the mouse lines, we designed site-specific genotyping assays for each copy in a known location, and we could differentiate heteroand homozygotes. In the case of U1 and U2, we could only detect the presence of the transgene, but we could not distinguish the hetero-and homozygotes; therefore, we bred them strictly with Sstr4 KO mice to obtain only heterozygous mice for testing. showing the full-length hSSTR4 coding sequence with up-and downstream regulatory elements (7.8 kb) as copied fragments and the neighboring genes (FOXA2 and THBD) (A). PB transposon vector carrying the upstream regulatory elements (4 kb) and human SSTR4 coding sequence followed by the P2A self-cleaving peptide sequence, luciferase and tdTomato coding sequences downstream SSTR4 regulatory elements (2.7 kb) and polyadenylation signal sequence (poly A). The entire transgenic cassette (12.7 kb) was flanked by the PB transposon inverted terminal repeats (ITR), insulators and Lox2272 Cre recombinase recognition sites (B).
The vector was verified by sequencing as successfully constructed before it was injected into 56 Sstr4 KO zygotes, along with the PB transposase mRNA (Figure 2A). After injection, the intact zygotes were implanted into three pseudo-pregnant Sstr4 KO mice ( Figure 2B). Sixteen mice were born in the F0 generation, out of which three female mice were verified by PCR carrying the PB transposon without the plasmid backbone ( Figure 2C). These transgene positive mice were crossbred with Sstr4 KO mice. Two females died in late pregnancy. The third female produced 8 offspring (F1 generation), but it also died during its second pregnancy ( Figure 2D). In this founder mouse, the PB transposon integration sites were located by ligation-mediated polymerase chain reaction (LM-PCR) ( Figure 2E). From the F1 generation onwards, all mouse lines carried a single copy of the transgene, and none of those lines manifest a harmful phenotype based on viability, fertility and behavior. There were no differences between the litter sizes of the mouse lines (WT: 6.2 ± 0.4, KO: 5.8 ± 0.3, Chr3: 6.5 ± 0.4, U1: 6.0 ± 0.4, and U2: 5.9 ± 0.4).

Integration Sites of the hSSTR4 Transgene
The LM-PCR detected multiple insertion loci of the transgene in the F0 founder female. By sequencing of the LM-PCR products, three distinct integration sites were identified in chromosomes 3, 10 and X (copies were named Chr3, Chr10 and ChrX, respectively). The transgene inserted in the chromosome 10 did not pass onto the F1 generation ( Figure 2). The transgene in the chromosome 3 was inserted between the Sis and Otol1 genes, coding on the negative strand of the chromosome (Figure 3). The PB transposase inserted the transposon while duplicating the TTAA recognition site, which started in the original position of 70,039,120 [47] (Figure 3). The transgene inserted in the chromosome 10 in the position of 67,335,940 [48], coding on the positive strand. The transgene inserted in the chromosome X at the position of 32,102,395 [49], coding on the positive strand. There were two mouse lines in which we were unable to locate the insertion site of the transgene (named U1 and U2) (   transgene. The transgene copies are named Chr3, Chr10 and ChrX (located in chromosomes 3, 10 and X, respectively) and U1 and U2 (the two copies in an unknown location).
To keep track of the transgene in the mouse lines, we designed site-specific genotyping assays for each copy in a known location, and we could differentiate hetero-and homozygotes. In the case of U1 and U2, we could only detect the presence of the transgene, but we could not distinguish the hetero-and homozygotes; therefore, we bred them strictly with Sstr4 KO mice to obtain only heterozygous mice for testing.

Distinct Expression Pattern of the hSSTR4-Related Luciferase by Luminescent In Vivo Optical Imaging in Various Mouse Lines
First, we characterized the expression of the transgene by measuring the signals of the co-expressed reporter fusion protein: the luminescence of the luciferase and the fluorescence of the tdTomato. Luciferase activity was visualized in the whole body ( Figure  4A) and quantified in regions of interest (ROI) in the area of the brain ( Figure 4B) by IVIS Lumina III. No luciferase activity was detected either in Sstr4 KO mice or in hemizygote male or homozygote female ChrX hSSTR4-positive mice. Therefore, we excluded the ChrX line from later research, since the transgene was not expressed. Copies in unknown loci (U1 and U2) showed similar expression patterns: weak signal in the extremities and tail, medium signal strength in the area of cerebrum and the caudal part of the brain and strongest signal in the area of olfactory bulb (OB). Chr3 mice showed the strongest signal in the area corresponding to the cerebral hemispheres. Chr3 homozygous mice had nearly exactly double the signal intensity compared to Chr3 heterozygous mice. Chr3 Het mice had significantly stronger signal than U1 and U2 (also heterozygous) mice ( Figure 4B). In the periphery, the luminescent signal was weaker and variable, with the highest levels in the lower abdomen and pelvis, regardless of the chromosomal location of the copy of the transgene. Fluorescence of tdTomato linked to hSSTR4 was not detectable in vivo by optical imaging (image is not shown).

Distinct Expression Pattern of the hSSTR4-Related Luciferase by Luminescent In Vivo Optical Imaging in Various Mouse Lines
First, we characterized the expression of the transgene by measuring the signals of the co-expressed reporter fusion protein: the luminescence of the luciferase and the fluorescence of the tdTomato. Luciferase activity was visualized in the whole body ( Figure 4A) and quantified in regions of interest (ROI) in the area of the brain ( Figure 4B) by IVIS Lumina III. No luciferase activity was detected either in Sstr4 KO mice or in hemizygote male or homozygote female ChrX hSSTR4-positive mice. Therefore, we excluded the ChrX line from later research, since the transgene was not expressed. Copies in unknown loci (U1 and U2) showed similar expression patterns: weak signal in the extremities and tail, medium signal strength in the area of cerebrum and the caudal part of the brain and strongest signal in the area of olfactory bulb (OB). Chr3 mice showed the strongest signal in the area corresponding to the cerebral hemispheres. Chr3 homozygous mice had nearly exactly double the signal intensity compared to Chr3 heterozygous mice. Chr3 Het mice had significantly stronger signal than U1 and U2 (also heterozygous) mice ( Figure 4B). In the periphery, the luminescent signal was weaker and variable, with the highest levels in the lower abdomen and pelvis, regardless of the chromosomal location of the copy of the transgene. Fluorescence of tdTomato linked to hSSTR4 was not detectable in vivo by optical imaging (image is not shown).

Figure 4.
In vivo bioluminescent imaging of the luciferase reporter protein co-expressed with the hSSTR4 gene. Representative images show the differences of expression pattern and luminescence intensity of luciferase in the different mouse lines (A). Scatter plot with bars show the means ± SEM with the individual data points of the luminescence intensity in equal size areas of the head corresponding to the brain (B). One-way ANOVA, * p < 0.0001, N = 6-19/genotype.

hSSTR4 Expression Level Assessed by RT-qPCR
In every inspected organ, with the exception of the lungs, Chr3 showed a higher expression of hSSTR4 than the mouse Sstr4 expression in the WT mice. Chr3 showed the highest hSSTR4 expression in the nervous system: cerebral cortex, olfactory bulb (OB), trigeminal ganglia (TG), cerebellum and brain stem, and also a relatively high expression in the epididymis. In comparison, the mouse Sstr4 expression in the WT mice was the highest in the cerebral cortex, lungs, OB, epididymis and TG but much lower in the brain In vivo bioluminescent imaging of the luciferase reporter protein co-expressed with the hSSTR4 gene. Representative images show the differences of expression pattern and luminescence intensity of luciferase in the different mouse lines (A). Scatter plot with bars show the means ± SEM with the individual data points of the luminescence intensity in equal size areas of the head corresponding to the brain (B). One-way ANOVA, * p < 0.0001, N = 6-19/genotype.

hSSTR4 Expression Level Assessed by RT-qPCR
In every inspected organ, with the exception of the lungs, Chr3 showed a higher expression of hSSTR4 than the mouse Sstr4 expression in the WT mice. Chr3 showed the highest hSSTR4 expression in the nervous system: cerebral cortex, olfactory bulb (OB), trigeminal ganglia (TG), cerebellum and brain stem, and also a relatively high expression in the epididymis. In comparison, the mouse Sstr4 expression in the WT mice was the highest in the cerebral cortex, lungs, OB, epididymis and TG but much lower in the brain stem and cerebellum than the humanized mice. Generally, U1 and U2 showed lower expression levels of hSSTR4 in every organ, with the exception of the lungs, compared to Chr3 mice. ( Figure 5). stem and cerebellum than the humanized mice. Generally, U1 and U2 showed lower expression levels of hSSTR4 in every organ, with the exception of the lungs, compared to Chr3 mice. ( Figure 5).

Figure 5.
Relative mouse Sstr4 and human SSTR4 mRNA expression levels in wild-type (WT) and humanized (Chr3, U1 and U2) mice, respectively. The diagram shows RT-qPCR results (2 −ΔCt ) relative to the beta actin (Actb) mRNA reference gene in the tested organs. Cortex, OB and TG stand for cerebral cortex, olfactory bulb and trigeminal ganglion, respectively. Each column shows the mean ± SEM. The significant differences between the WT and Chr3 mice are indicated with an asterisk above the Chr3 column. Kruskal-Wallis test with Dunn's post-test; * p < 0.01; N = 3-7/genotype.

Expression of hSSTR4 mRNA in Different Types of Neurons in the Brain
Since the hSSTR4-related tdTomato fluorescence was not detectable in native brain sections either (data not shown), and there is no reliably specific anti-SST4 antibody on the market, in situ hybridization of the hSSTR4 mRNA was performed to explore its expression pattern. In brain samples of the hSSTR4 Chr3 homozygous mice, the hSSTR4 transcript showed a moderate expression level. Within the primary somatosensory cortex (S1), the highest level of expression was found in layers II and III ( Figure 6A,B). The highest hSSTR4 expression was detected in the CA2 field of the hippocampus and in the piriform cortex (Figure 7). Other areas of the brain, such as the granular layer of the OB, Figure 5. Relative mouse Sstr4 and human SSTR4 mRNA expression levels in wild-type (WT) and humanized (Chr3, U1 and U2) mice, respectively. The diagram shows RT-qPCR results (2 −∆Ct ) relative to the beta actin (Actb) mRNA reference gene in the tested organs. Cortex, OB and TG stand for cerebral cortex, olfactory bulb and trigeminal ganglion, respectively. Each column shows the mean ± SEM. The significant differences between the WT and Chr3 mice are indicated with an asterisk above the Chr3 column. Kruskal-Wallis test with Dunn's post-test; * p < 0.01; N = 3-7/genotype.

Expression of hSSTR4 mRNA in Different Types of Neurons in the Brain
Since the hSSTR4-related tdTomato fluorescence was not detectable in native brain sections either (data not shown), and there is no reliably specific anti-SST 4 antibody on the market, in situ hybridization of the hSSTR4 mRNA was performed to explore its expression pattern. In brain samples of the hSSTR4 Chr3 homozygous mice, the hSSTR4 transcript showed a moderate expression level. Within the primary somatosensory cortex (S1), the highest level of expression was found in layers II and III ( Figure 6A,B). The highest hSSTR4 expression was detected in the CA2 field of the hippocampus and in the piriform cortex ( Figure 7). Other areas of the brain, such as the granular layer of the OB, the prelimbic cortex, the basolateral (BLA) and the basomedial (BMA) nucleus of the amygdala, showed low, but still considerable hSSTR4 expression levels ( Figure 8).
hSSTR4 was predominantly localized on glutamatergic excitatory neurons (Vglut1) with inconsistent cases in the GABAergic interneurons (Gad1) in the primary somatosensory cortex (Figure 6), piriform cortex ( Figure 7C), prelimbic cortex ( Figure 8B), BLA ( Figure 8C) and BMA ( Figure 8D). In the hippocampus, CA1 and CA2 hSSTR4 were localized in the glutamatergic excitatory neurons ( Figure 7A,B). In the granular layer of the OB, hSSTR4 transcripts were detected in the GABAergic interneurons ( Figure 8A). See the control samples in Appendix A ( Figure A1). the prelimbic cortex, the basolateral (BLA) and the basomedial (BMA) nucleus of the amygdala, showed low, but still considerable hSSTR4 expression levels ( Figure 8). hSSTR4 was predominantly localized on glutamatergic excitatory neurons (Vglut1) with inconsistent cases in the GABAergic interneurons (Gad1) in the primary somatosensory cortex (Figure 6), piriform cortex ( Figure 7C), prelimbic cortex ( Figure 8B), BLA ( Figure  8C) and BMA ( Figure 8D). In the hippocampus, CA1 and CA2 hSSTR4 were localized in the glutamatergic excitatory neurons ( Figure 7A,B). In the granular layer of the OB, hSSTR4 transcripts were detected in the GABAergic interneurons ( Figure 8A). See the control samples in Appendix A ( Figure A1).

Discussion
The main impacts of the present work are that we successfully generated the first hSSTR4 transgenic mouse line via random insertion of a PB transposon vector and characterized the receptor-expressing neurons in pain and mood regulation-related brain regions. These mice can be useful tools for preclinical research of the SST 4 receptor, which is a promising novel target for analgesic and antidepressant drug development [2,22,23].
We chose random insertion instead of the popular knock-in approach, because we aimed to avoid the influence of mouse regulatory elements of the Sstr4 gene on the transgene. We used a transposon vector with the intact human regulatory elements flanked by insulator regions to resemble the human receptor expression pattern. However, the disadvantage of this technique is that the mapping of the integration sites can be problematic, and the insert may disrupt mouse genes [50,51].
The random insertion of the PB transposon resulted in multiple integration sites in the F0 mouse generation. We successfully located three copies of the transgene by LM-PCR [52]: Chr3, Chr10 and ChrX, but the insertion sites of two copies (U1 and U2) are still unknown. The unsuccessful attempts to locate the integration sites of U1 and U2 suggest that they were inserted in repeat regions of the genome that makes mapping of the transgene difficult. Due to the known location of the transgene in the Chr3 mouse line, we could design site-specific genotyping assay to distinguish hetero-and homozygous mice.
All three transgenic mice in the F0 generation experienced complications during pregnancy and delivery and eventually died. This might have been due to the multicopy insertion of the transgene and the consequent overexpression of SST 4 , because this problem never occurred again in the offspring carrying a single copy. This observation also supports a role of SST 4 in embryonal growth regulation, since it was demonstrated to be the predominant somatostatin receptor in the human placenta [53,54].
Bioluminescent in vivo optical imaging showed hSSTR4-linked luciferase expression pattern in different organs, and quantification showed that the luminescent signal was the strongest in the area corresponding to the brain. Chr3 mice showed a high expression in the area of the cerebrum, whereas U1 and U2 showed a lower expression here but a higher expression in the area of the OB and the caudal part of the brain. RT-qPCR supported these results, as it also showed the highest hSSTR4 expressions in the cerebral cortex and the OB. The mouse Sstr4 expression was somewhat lower but similar to the hSSTR4 expression in Chr3 mice, with the exception of the lungs, where it was much higher, and the cerebellum and brain stem, where it was much lower. These findings are in agreement with previous data obtained in large-scale expression studies of both the human and mouse receptors [46,[54][55][56]. In both luciferase IVIS and RT-qPCR, U1 and U2 showed some similarities in hSSTR4 expression for both the pattern and level, suggesting that these two mouse lines carry a copy in the same integration site, but this needs further investigation. Each mouse showed individually different luciferase activity for both the intensity and pattern in the areas of the abdomen and the pelvis. The databases demonstrate variable SST 4 expression levels (from not detected to moderate) in both human and mouse gastrointestinal and reproductive systems [57][58][59][60][61]. The Chr3 RT-qPCR results showing the relatively high hSSTR4 expression in the epididymis support this data, but the lower expression in the stomach and the intestine does not. Chr3 had the strongest luciferase signal in the area of the brain (roughly three times as much as observed in U1 and U2), and unlike U1 and U2, we could distinguish and compare the hetero-and homozygous mice. This showed a strong positive association: the double the gene, the double the signal intensity. Therefore, we selected Chr3 as the most useful mouse line and characterized the hSSTR4 expression by RNAscope.
The tdTomato fluorescence was not detectable in any of the mouse lines either in vivo or in the histological sections, probably due to the low expression level of the hSSTR4 transgene. TdTomato is usually driven by a strong viral promoter like the human cytomegalovirus or the promoter of a mammalian housekeeping gene like elongation factor-1 alpha, to be expressed at high level for a strong fluorescent signal [62]. Furthermore, while the tdTomato has been considered more tolerant of N-terminal fusion than the mRFP1 it was derived from [63], we observed its fluorescence to be greatly diminished in the luciferase-tdTomato fusion protein compared to the native tdTomato protein (data not shown), probably due to the disrupted folding or tetramerization of the tdTomato [64,65].
The hSSTR4 had the most prominent signal in the hippocampus (CA1 and CA2) and the cerebral cortex (Pir, S1 and PrL), which corresponds to the mouse and human databases [57][58][59][60][61]. RNAscope in the brain of the Chr3 mice shows hSSTR4 predominantly to be expressed in Vglut1-positive glutamatergic excitatory neurons similarly to the Sstr4 receptor in wild-type mice, although at a visibly lower expression level. hSSTR4 was also expressed in GABAergic interneurons in the same regions, whereas, previously, the mouse Sstr4 mRNA was detected in GABAergic neurons only in the core of the central amygdala. In S1, hSSTR4 had a higher expression in layer V than in layers II-IV, as opposed to the mouse Sstr4 expression [46]. In a previous study, in the OB of the wild-type mice, Sstr4 was expressed in the glomerular layer but not in the granular cell layer [66], whereas, in transgenic mice, the hSSTR4 was expressed mostly in the granular layer of the OB.
Differences in the expression pattern between human SSTR4 and mouse Sstr4 might be due to species differences but can also result from limitations of the humanized mouse model, such as the positional effect of the integration site [51]. Therefore, these differences need to be further investigated.
We concluded that the Chr3 hSSTR4 mouse line showed measurable hSSTR4 expression, mainly in excitatory glutamatergic neurons of pain-and mood regulation-related brain regions, with several similarities and, also, some differences compared to the mouse Sstr4 expression. Therefore, after confirming the receptor function, this transgenic mouse line can be a suitable translational research tool to determine the potential of SST 4 as an analgesic, antidepressant and anti-inflammatory drug target and to test the SST 4 agonist candidates during preclinical development.

PiggyBac Transposon Vector
Vector pPBC2 was constructed as follows: The 4 kb sequence upstream of the hSSTR4 coding region containing the promoter and putative regulatory sequences was obtained in two PCR reactions on human genomic DNA. The first segment was amplified by the forward primer 5 -ATC CTC ATT CAC TAT CCT GGG AAG T-3 and reverse primer 5 -CCT GGA ATC TTT CCT GTG CCT ACT T-3 resulting in a 2082-bp-long fragment. The second segment containing the beginning of the hSSTR4 coding region, and the immediate upstream sequence was amplified by using forward primer 5 TCC TGG AAG CAC TAG CTG TTT ATC A-3 and reverse primer 5 -TTC ACC AGC GTC TTC TGT CTC ACC-3 , producing a 2580-bp-long fragment. The two PCR fragments were cloned together at the SphI restriction site in the overlapping section, and a XhoI-SmaI fragment of it was incorporated into the construct containing the beginning of the SSTR4 coding region and 3968 bp upstream sequence. The rest of the hSSTR4 coding sequence was cloned from vector pcDNA3.1(+)/SSTR4 (Cat# SSTR400000; Guthrie Research Institute, Sayre, PA, USA) and fused to the previously described fragment at the unique SmaI site in the hSSTR4 coding sequence. In the final construct, the coding region contains four singlenucleotide polymorphisms (SNP) compared to the human SSTR4 reference sequence [67]. These high-frequency SNPs are rs3746726 850T > G, rs2567609 897T > C, rs3746728 924C > T and rs2567608 962T > C. The 2645-bp-long downstream genomic region behind the hSSTR4 coding sequence was obtained by genomic PCR using forward primer 5 -GGA GCC CTT CCC CTA CCC A-3 and reverse primer 5 -TGG GTA GGG GAA GGG CTC C-3 . The fragment coding the luciferase-tdTomato marker protein was cloned from vector pcDNA(+)/Luc2 = tdT (Cat#32904; Addgene, Watertown, MA, USA). The sequence coding the P2A peptide was constructed from oligonucleotides and inserted between the hSSTR4 and luciferase-tdTomato sequences. Lox2273 sites and the artificial polyA site was also assembled from oligonucleotides and inserted into the appropriate positions. Finally, the whole construct was inserted into the pB007 PiggyBac vector (Ref# SPB-007; Transposagen, Lexington, KY, USA) to obtain the pPBC2 vector that was used for transgenesis.

Animals
B6.129P2-Sstr4 tm1Szo (Sstr4 KO) mice [2,22,23] were used for the transgenesis (both the zygotes and the pseudo-pregnant females), for the early breeding of transgenic mice and, later, as control animals in experiments. Distinct mouse lines were created by breeding, each containing a single copy of the transgene. The proposed name of the humanized SSTR4 expressing mouse strain is B6.129P2-Sstr4 tm1Szo -TgTn(pb-SSTR4-P2A-luc-tdTomato)1Sazo.
Animals were bred and kept in the animal house of the Department of Pharmacology and Pharmacotherapy, University of Pécs Medical School at a temperature of 22 ± 2 • C and a 12-h light-dark cycle. Standard rodent chow and water were provided ad libitum.

Transgenesis
The transgenesis was made in commission by BioTalentum Ltd. (Gödöllő, Hungary). The hSSTR4 transgene without the plasmid backbone integrated into only 3 female mice from the F0 generation, and all of them were backcrossed with Sstr4 KO males.

Ligation-Mediated PCR
We followed the protocol and Y-linker sequences from Bryda et al. [52]. Genomic DNA was extracted from tail biopsies using the Thermo Scientific Phire Tissue Direct PCR Master Mix kit (Thermo Fisher Scientific, Waltham, MA, USA). Digestion was performed with different 3 overhang creator restriction endonucleases separately: TaiI, PstI and HhaI. Three transgene specific nested primers were designed to both 5 and 3 end regions of the transgene using Primer-BLAST (NCBI, Rockville Pike, Bethesda MD, USA). The nested primers were oriented to the outward of the transgene, where the Y-linker is expected to be attached, and the primers were arranged accordingly (primer 1 is furthest from the end of the transgene, and primer 3 is the closest).  [52]. The Y-linker-specific nested PCR primers: Y-linker primer D: GCA AAC GAT AAA TGC GAG GAC GGT and Y-linker primer G: ATG CGA GGA CGG TAC AGG CCG ACC. All primers and the Y-linker were synthesized by Integrated DNA Technologies (IDT; Coralville, IA, USA). The Y-linker was prepared by the annealing of Y-linker A and Y-linker E. The first (single round) PCR was performed with digested DNA, Taq DNA polymerase enzyme (5 overhang synthesizing tendency) and transgene specific primer 1 (LM-V1 and LM-V1B in separate reactions). The Y-linker was ligated with the product of the first PCR by T4 DNA ligase and then was amplified by 2 consecutive PCRs with nested primer pairs. The second PCR was made with the product of ligation, Y-linker primer D and transgene specific primer 2 (LM-V2 or LM-V2B, matching to the primer 1 used in the first PCR) and the third PCR was made with the product of the second PCR, Y-linker primer G and transgene specific primer 3 (LM-V3 or LM-V3B, matching to the primer 1 and 2 used in the previous PCRs). Each was made with Phire Tissue Direct PCR Master Mix. Products of the third PCR were separated by gel electrophoresis, and all detected bands were isolated using the GeneJET Gel Extraction Kit (Thermo Fisher Scientific, Waltham, MA, USA). The nucleotide sequence of each product of the third PCR was acquired by Sanger sequencing in the commissioned BIOMI Ltd. (Gödöllő, Hungary). The acquired sequences were analyzed by National Center for Biotechnology Information (NCBI) Nucleotide-BLAST. For verification of the identified loci, mouse chromosome specific (near the insertion site of the transgene) primers were designed to be paired with a LM-V3 transgene specific primer: ChrXpr1: AAC TCC TTT ACC CGC TTG CTC for chromosome X, Chr3pr2: CTG GTT CCG AGT CTC TGA GG for chromosome 3 and Chr10pr2: ATA ATG CCC CTG GCA TAG CTT TC for chromosome 10. Chr3 hSSTR4 mice are since genotyped routinely by LM-V3 forward primer, Chr3pr2 reverse primer and a secondary forward primer Chr3pr3: TCA GGA GCA AGA GAG GAA GA, resulting in PCR products in the size of 527 bp for wild-type chromosomes and 683 bp for the inserted hSSTR4 transgene, allowing the identification of Chr3 homozygotes and heterozygotes ( Figure 3).

Detecting hSSTR4 Expression by In Vivo Optical Imaging of the Luciferase Enzyme and tdTomato
For the in vivo imaging of luciferase, 8-10-week-old male animals were injected with 300 mg/kg D-luciferin sodium salt (Goldbio, St. Louis, MO, USA) intraperitoneally (i.p.) and then anaesthetized with ketamine-xylazine (100 and 5 mg/kg i.p.). The fur was removed with a fine electrical shaver. Bioluminescent imaging was performed 30 min after D-luciferin administration with the IVIS Lumina III imaging system (PerkinElmer, Waltham, MA, USA) with the following settings: exposure time 5 min and, binning 4. The bioluminescent signal of the brain was quantified as a total flux (photons/s) in equal-size regions of interest (ROI) corresponding to the top of the skull. Fluorescent optical imaging of tdTomato expression was also performed with the IVIS Lumina III imaging system. For tdTomato detection, excitation filters of 500, 520, 540 and 560 nm for spectral unmixing and an emission filter of 620 nm, auto exposure and a binning of 2 were used. After imaging, animals were placed onto a heating pad and monitored until they recovered from anesthesia.

Investigating hSSTR4-Linked tdTomato Expression in the Mouse Brain by Confocal Microscopy
After luminescent optical imaging, the animals were deeply anesthetized with an overdose of urethane (2.4 g/kg i.p.) and perfused transcardially with 30 mL of 4% paraformaldehyde in Millonig's phosphate buffer. Dissected brains were postfixed for 24 h at room temperature (RT), rinsed in 1x phosphate-buffered saline (PBS) and sectioned (by 30 µm) using a vibrating microtome (VT1000S, Leica Biosystems, Wetzlar, Germany). Sections were mounted on Superfrost Ultra Plus slides (Thermo Fisher Scientific, Waltham, MA, USA), air-dried for 3 h at RT and the sections were counterstained with 4 ,6-diamidino-2phenylindole (DAPI) and mounted with ProLong Diamond Antifade Mountant (Thermo Fisher Scientific, Waltham, MA, USA) for confocal imaging.
Fluorescent images of PrL, BLA, S1, CA1, CA2 and Pir, according to Paxinos and Franklin [48], were acquired using an Olympus Fluoview FV-1000 laser scanning confocal microscope and FluoView FV-1000S-IX81 image acquisition software system (Olympus, Tokyo, Japan). The confocal aperture was set to 80 µm. The analog sequential scanning was performed using a 40x objective lens (NA: 0.75). The optical thickness was set to 1 µm, and the resolution was 1024 × 1024 pixels. The excitation time was set to 4 µs per pixel. Virtual colors were selected to depict fluorescent signals: blue for DAPI and red for tdTomato).
Images of the two respective channels were stored both individually and superimposed to evaluate the colocalization of fluorescent signals.

Measuring Organ-Specific hSSTR4 Expression by RT-qPCR
Total RNA was extracted from WT (C57BL/6), Sstr4 KO, Chr3, U1 and U2 8-10week-old male mice from the following 14 mouse organs: bladder, brain stem, cerebellum, cerebral cortex, epididymis, heart, ileum, kidney, liver, lungs, OB, spleen, stomach and TG. TRI Reagent ( These primers were tested on KO mice (data not shown) and RT-qPCR amplification and the melting curve analysis indicated that both the human SSTR4 and mouse Sstr4 primers are specific to their respective genes. Water control was used to check the reactions for contamination. Real-time qPCR was performed under the following conditions: 95 • C for 10 min, followed by 40 cycles of 95 • C for 30 s, 60 • C for 30 s and 72 • C for 1 min. Each reaction was carried out in a duplicate. Signal specificity was ensured by melt curve analysis. Relative expression ratios were calculated using the ∆Ct method and linearized by 2ˆ-∆Ct, in which the hSSTR4 expression level was compared to the Actb reference gene.
RNAscope assay was performed on 30-µm-thick vibratome-sliced brain sections using RNAscope Multiplex Fluorescent Reagent Kit v2 (Advanced Cell Diagnostics, Newark, CA, USA), according to the manufacturer's protocols. Tissue pretreatment was performed by treatment with 1 v/v% H 2 O 2 solution in PBS for 30 min. After PBS washes, sections were mounted on Superfrost Ultra Plus slides (Thermo Fisher Scientific, Waltham, MA, USA). Sections were air-dried for 3 h at RT and incubated at 60 • C for 60 min. After 2 × 10-min washes in Milli-Q (MQ) water, slides were incubated in 10% neutral buffered formalin solution (NBF, Merck KGaA) at 4 • C for 2 min. After 3 × 10 min MQ water rinses, sections were digested in proteinase K solution at 37 • C for 15 min (0.01 mg/ml proteinase K (EO0491, Thermo Fisher Scientific, Waltham, MA, USA) in 0.1 M Tris/HCl, pH = 8 and 0.05 M EDTA, pH = 8 buffer. After rinsing in MQ water, slides were treated with 10% NBF at 4 • C for 2 min, followed by 3 × 10-min washes in MQ water. Sections were hybridized with probes specific to human SSTR4 and mouse Sstr4, Vglut1 and Gad1, in parallel with RNAscope 3-plex mouse positive and negative control probes. Signal amplification, channel development and mounting were performed according to the manufacturer's protocols. Sections were counterstained with 4 ,6-diamidino-2-phenylindole (DAPI) and mounted with ProLong Diamond Antifade Mountant (Thermo Fisher Scientific, Waltham, MA, USA) for confocal imaging. Fluorescent images of PrL, BLA, S1, CA1, CA2 and Pir, according to Paxinos and Franklin [68], were acquired using an Olympus Fluoview FV-1000 laser scanning confocal microscope and FluoView FV-1000S-IX81 image acquisition software system (Olympus, Tokyo, Japan). The confocal aperture was set to 80 µm. The analog sequential scanning was performed using a 40x objective lens (NA: 0.75). The optical thickness was set to 1 µm, and the resolution was 1024 × 1024 pixels. The excitation time was set to 4 µs per pixel. Virtual colors were selected to depict the fluorescent signals: blue for DAPI, green for fluorescein (Vglut1 mRNA), red for Cyanine 3 (hSSTR4 and mSstr4 mRNA) and white for Cyanine 5 (Gad1 mRNA). Images of the respective four channels were stored both individually and superimposed to evaluate the co-localization of fluorescent signals.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.

Acknowledgments:
The authors are thankful to Miklós Kecskés for his useful suggestions for data interpretation and further functional studies, Nikolett Szentes for collecting and processing mouse organs for RT-qPCR, Lilla Draskóczi for processing mouse organs for RT-qPCR, and Tünde Bíró-Sütő for processing the mouse brain samples for microscopy. The in vivo imaging experiments were performed in the Small Animal Imaging Core Facility of the Szentágothai Research Centre, University of Pécs, Hungary.

Conflicts of Interest:
We declare no conflict of interest. Z.H. and E.P. are the founders of PharmInVivo Ltd., Pécs, Hungary. Z.H. and E.P. are stakeholders of ALGONIST Biotechnologies GmbH, Wien, Austria. The funders had no role in the design of the study; in the collection, analyses or interpretation of data; in the writing of the manuscript or in the decision to publish the results.