Expression Of lacZ Reporter Gene Research Articles

Nhlrc2 is crucial during mouse gastrulation.

SummaryThe loss of NHL repeat containing 2 (Nhlrc2) leads to early embryonic lethality in mice, but the exact timing is currently unknown. In this study, we determined the time of lethality for Nhlrc2 knockout (KO), C57BL/6NCrl‐Nhlrc2 tm1a(KOMP)Wtsi/Oulu, embryos and the in situ expression pattern of Nhlrc2 based on LacZ reporter gene expression during this period. Nhlrc2 KO preimplantation mouse embryos developed normally after in vitro fertilization. Embryonic stem (ES) cells established from KO blastocysts proliferated normally despite a complete loss of the NHLRC2 protein. Nhlrc2 KO embryos from timed matings implanted and were indistinguishable from their wildtype littermates on embryonic day (E) 6.5. On E7.5, Nhlrc2 KO embryo development was arrested, and on E8.5, only 6% of the genotyped embryos were homozygous for the Nhlrc2 tm1a(KOMP)Wtsi allele. Nhlrc2 KO E8.5 embryos showed limited embryonic or extraembryonic tissue differentiation and remained at the cylinder stage. Nhlrc2 expression was ubiquitous but strongest in the epiblast/ectoderm and extraembryonic ectoderm on E6.5 and E7.5. NHLRC2 is essential for early postimplantation development, and its loss leads to failed gastrulation and amniotic folding in mice. Future studies on the evolutionarily conserved NHLRC2 will provide new insights into the molecular pathways involved in the early steps of postimplantation development.

Sex Differences in Androgen Receptor Enhancer Activity and Expression during Mus musculus Embryonic Development

Study objectiveThe objective of this study is to compare the sex‐specific enhancer function of a cis‐regulatory element during mammary gland development to the expression of the androgen receptor (AR) in mammary tissues.IntroductionConserved noncoding elements regulate gene expression during mammalian development with remarkable precision. AR is critical for the development of male specific phenotypes in many tissues, yet little is known regarding the tissue‐specific regulation of AR expression. We have previously identified a 7.7kb sequence near the AR gene that activates lacZ reporter gene expression in prenatal and postnatal mice. Embryologically, the enhancer drives consistent lacZ expression in developing hair follicles and the genital tubercle. However, expression in the mammary ridge/glands occurs in only half of the transgenic embryos produced. Each of these structures are known sites of expression of AR or are affected by androgens during growth and development. We hypothesize that the variability in this enhancer function during mammary gland development may be sex dependent.MethodsWe utilized a previously established transgenic mouse line containing the 7.7kb AR enhancer mouse sequence linked to a basal promoter driving lacZ. Mice were collected and stained during different stages of embryonic development to visualize localization of AR enhancer activity. The sex of the embryos were identified by PCR amplification of the Sry gene. We compared enhancer driven lacZ expression to endogenous AR expression determined by in situ hybridization (ISH) in mouse embryos.ResultsWe have surveyed lacZ expression at E15.5 (embryonic day) and E16.5. Despite consistent expression in other tissues, mammary gland expression of lacZ occurs only in female embryos. No mammary associated lacZ expression is visible in males. These expression results are consistent with AR gene expression as detected by ISH.ConclusionWe show that there are sex differences in enhancer activity of AR during mouse development. AR is crucial for inhibiting the development of female‐specific traits as well as promoting the male phenotype. The lack of lacZ expression in male embryos suggests the inhibitory effects of androgens have been sufficient to either eliminate the mammary associated cell types that normally express AR or the expression of an upstream trans‐acting factor that activates this AR enhancer. This mouse line will be useful for tracing developmental progression of AR expressing tissues and signalling during mammalian integumentary development as well as the development of sexually dimorphic characteristics.Support or Funding InformationFunding provided by NSF IOS‐1656315 & NSF BCS‐1638812 and PCOM's Center for Chronic Disorders of Aging.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

In vivo target exploration of apidaecin based on Acquired Resistance induced by Gene Overexpression (ARGO assay)

Identifying the target molecules of antimicrobial agents is essential for assessing their mode of action. Here, we propose Acquired Resistance induced by Gene Overexpression (ARGO) as a novel in vivo approach for exploring target proteins of antimicrobial agents. The principle of the method is based on the fact that overexpression of the expected target protein leads to reduced sensitivity to the antimicrobial agent. We applied this approach to identify target proteins of the antimicrobial peptide apidaecin, which is specifically effective against Gram-negative bacteria. To this end, a set of overexpression Escherichia coli clones was tested, and peptide chain release factor 1, which directs the termination of translation, was found as a candidate, suggesting that apidaecin inhibits the termination step of translation. This finding was confirmed in vivo and in vitro by evaluating the inhibitory activity of apidaecin towards lacZ reporter gene expression, which is tightly dependent on its stop codon. The results of this study demonstrate that apidaecin exerts its antimicrobial effects partly by inhibiting release factors.

Comparison of lacZ reporter gene expression in gilthead sea bream (Sparus aurata) following oral or intramuscular administration of plasmid DNA in chitosan nanoparticles

The development of non-parenteral delivery systems allowing efficient intestinal absorption in fish is a current research topic drawing much interest. Different aspects would benefit from such oral delivery, especially those related to immunization, owing to the convenience of this route in terms of time saving, safety, and lack of stressful effects among others. In the last years, DNA vaccines are evidencing their suitability for fish immunization, although commercial preparations currently available require parenteral administration. In this regard, this work was aimed at assessing a chitosan-based oral delivery system for plasmid DNA (pDNA) in fish. Plasmid pCMVβ was encapsulated in chitosan nanoparticles with the aim of evaluating the ability of inert capsules to retain and protect pDNA from in vitro conditions mimicking the fish gut environment. Furthermore, the feasibility of in vivo oral gene delivery was also assessed in fish by administrating pDNA nanoparticles comparing oral and intramuscular routes. The results indicated that lacZ gene expression (β-galactosidase activity) could be detected in fish tissues following both oral and i.m. administration of pDNA-chitosan nanoparticles up to 60days. However, organ distribution of the reporter gene expression was more evident after oral (β-gal activity measured in gut, liver and muscle) than after parenteral administration (mostly restricted to adjacent tissues). The results suggest that chitosan nanoparticles enabled efficient oral delivery of pDNA, followed by organ migration and further sustained expression of the reporter gene encoding for β-galactosidase. The intensity of such expression was similar to that obtained by intramuscular injection, a fact that might suppose valuable practical applications in terms of on-farm administration of DNA-based vaccines to fish.

Comments on Methods to Suppress Endogenous β-Galactosidase Activity in Mouse Tissues Expressing the LacZ Reporter Gene.

The Escherichia coli LacZ gene (encoding β-galactosidase) is a widely used reporter for gene regulation analysis in transgenic mice. Determination of β-galactosidase activity is classically performed using 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside/ferri-/ferrocyanide (X-Gal/FeCN) histochemistry. Uncertainty about the origin of the β-galactosidase signal is encountered in tissues containing high levels of endogenous β-galactosidase. Here, we show that reliable results can nevertheless be obtained in these tissues by performing the histochemical reaction under slightly basic pH conditions (pH 8-9). We further demonstrate that in this context, analysis of tissue sections may be advantageous over that of conventional whole-mount tissues because poor dye penetration and remaining tissue acidity are avoided in tissue sections. We also recommend that bacterial debris should always be carefully removed from the luminal surface of gastrointestinal tract specimens unless staining of resident microflora is deliberately used as an internal positive control in the assay. Finally, we show that 6-chloro-3-indolyl-β-d-galactopyranoside with nitrotetrazolium blue chloride works well as an alternative chromogenic substrate for visualizing LacZ reporter gene expression in cryostat sections. Its use in high endogenous β-galactosidase-expressing organs is superior over the use of X-Gal/FeCN at slightly basic pH conditions.

Ligand determinants of nisin for its induction activity

Nisin has been widely used in the food industry as a safe and natural preservative and has the potential to be used as a biomedicine. Improving nisin production is important for its enormous applications. Nisin A is produced in Lactococcus lactis and its biosynthesis is induced through the regulation of the 2-component system NisKR. In this study, alanine-scanning mutagenesis was applied to study the key structure or AA in nisin for inducing the 2-component system NisKR to regulate downstream gene expression. Assay of β-galactosidase activity revealed that either ring A or ring B was necessary for nisin to induce lacZ reporter gene expression. A substituted first ring formed by Thr2 and Cys7 in S3A instead of ring A (formed by Ser3 and Cys7) fully retained nisin induction activity. Mutation of cationic AA and addition of cationic ions hardly affected nisin induction activity. These results demonstrated that the N-terminal ring structures in nisin were involved in activating NisKR to act as an inducing molecule, whereas the electrostatic force might not contribute to this process. In addition, 2 specific residues were revealed to have potential for improving both nisin induction and antimicrobial activity, which might be used for increasing nisin production.

Prediction and identification of an acid-inducible promoter from Lactococcus lactis ssp. cremoris MG1363

Using a promoter online prediction system, the gene sequence of GroEL protein from Lactococcus lactis ssp. cremoris MG1363 was analysed and a putative promoter, designated as gro, was further studied. Under acidic conditions, gro showed preliminary promoter function by driving the expression of lacZ reporter gene in Escherichia coli DH5α. In addition, gro drove the gene expression of GroEL and green fluorescent protein. In this paper, an acid-inducible promoter was successfully identified. This promoter has a wide prospect in the field of biotechnology.

A distinct regulatory region of the Bmp5 locus activates gene expression following adult bone fracture or soft tissue injury

Bone morphogenetic proteins (BMPs) are key signaling molecules required for normal development of bones and other tissues. Previous studies have shown that null mutations in the mouse Bmp5 gene alter the size, shape and number of multiple bone and cartilage structures during development. Bmp5 mutations also delay healing of rib fractures in adult mutants, suggesting that the same signals used to pattern embryonic bone and cartilage are also reused during skeletal regeneration and repair. Despite intense interest in BMPs as agents for stimulating bone formation in clinical applications, little is known about the regulatory elements that control developmental or injury-induced BMP expression. To compare the DNA sequences that activate gene expression during embryonic bone formation and following acute injuries in adult animals, we assayed regions surrounding the Bmp5 gene for their ability to stimulate lacZ reporter gene expression in transgenic mice. Multiple genomic fragments, distributed across the Bmp5 locus, collectively coordinate expression in discrete anatomic domains during normal development, including in embryonic ribs. In contrast, a distinct regulatory region activated expression following rib fracture in adult animals. The same injury control region triggered gene expression in mesenchymal cells following tibia fracture, in migrating keratinocytes following dorsal skin wounding, and in regenerating epithelial cells following lung injury. The Bmp5 gene thus contains an “injury response” control region that is distinct from embryonic enhancers, and that is activated by multiple types of injury in adult animals.

Functional characterization and expression profiling of a DREB2-type gene from lettuce (Lactuca sativa L.)

Dehydration responsive element binding factors (DREBs) play important roles in plant responses to abiotic stress. In this study, a DREB2-type gene, LsDREB2A, was isolated from lettuce (Lactuca sativa L.) and characterized. LsDREB2A encoded a protein of 322 amino acid residues with the conserved apetala 2 domain and was classified into the A2 subgroup of DREBs. LsDREB2A has no sequence rich in proline, glutamic acid, serine, and threonine to trigger protein degradation. Southern blot analysis indicated that there could be more than ten DREB homologs in lettuce. A gel shift assay indicated that LsDREB2A could specifically recognize the dehydration responsive element (DRE) sequence in vitro. In a yeast one-hybrid assay, LsDREB2A bound specifically to the DRE sequence and activated the expression of both His3 and LacZ reporter genes. A transient expression experiment in lettuce protoplasts showed that LsDREB2A was localized to the nucleus. These results suggest that LsDREB2A might function as a transcription factor. Quantitative real-time polymerase chain reaction revealed that the expression of LsDREB2A was significantly increased by hyperosmotic and high salinity treatment, but not cold, heat, nor abscisic acid (ABA) treatment. Overexpression of LsDREB2A increased the tolerance of salt stress in transgenic plants. These results suggested that LsDREB2A plays an important role during high salinity stress in an ABA-independent pathway.

Targeting gene expression to the female larval fat body of transgenic Aedes aegypti mosquitoes

As the fat body is a critical tissue for mosquito development, metamorphosis, immune and reproductive system function, the characterization of regulatory modules targeting gene expression to the female mosquito fat body at distinct life stages is much needed for multiple, varied strategies for controlling vector-borne diseases such as dengue and malaria. The hexameric storage protein, Hexamerin-1.2, of the mosquito Aedes atropalpus is female-specific and uniquely expressed in the fat body of fourth instar larvae and young adults. We have identified in the Hex-1.2 gene, a short regulatory module that directs female-, tissue-, and stage-specific lacZ reporter gene expression using a heterologous promoter in transgenic lines of the dengue vector Aedes aegypti. Male transgenic larvae and pupae of one line expressed no Escherichia coli β-galactosidase or transgene product; in two other lines reporter gene activity was highly female-biased. All transgenic lines expressed the reporter only in the fat body; however, lacZ mRNA levels were no different in males and females at any stage examined, suggesting that the gene regulatory module drives female-specific expression by post-transcriptional regulation in the heterologous mosquito. This regulatory element from the Hex-1.2 gene thus provides a new molecular tool for transgenic mosquito control as well as functional genetic analysis in aedine mosquitoes.

Development and characterization of a vector set with regulated promoters for systematic metabolic engineering in Saccharomyces cerevisiae

A set of vectors was constructed that enable combined and systematic testing of metabolic pathway genes in Saccharomyces cerevisiae. The vectors are available as CEN/ARS and 2 µ-based plasmids with a choice of three inducible promoters, P(GAL1) , P(CUP1) and P(ADH2) . These features offer control over the initiation and level of gene expression. In addition, the vectors can be used as templates to generate PCR fragments for targeted chromosomal integration of gene expression cassettes. Selection markers are flanked by loxP elements to allow efficient CreA-mediated marker removal and recycling after genomic integration. For each promoter, expression of a bacterial lacZ reporter gene was characterized from plasmid-based and integrated chromosomal cassettes, and compared to that of the glycolytic P(PGK1) promoter. Plasmid stabilities were also determined. The promoters showed distinct activity profiles useful for modulating expression of metabolic pathway genes. This series of plasmids with inducible promoters extends our previous vector set carrying the constitutive promoters P(PGK1) , P(TEF1) and P(HXT7-391) .

The regulatory factor PcRFX1 controls the expression of the three genes of β-lactam biosynthesis in Penicillium chrysogenum

Penicillin biosynthesis is subjected to a complex regulatory network of signalling molecules that may serve as model for other secondary metabolites. The information provided by the new “omics” era about Penicillium chrysogenum and the advances in the knowledge of molecular mechanisms responsible for improved productivity, make this fungus an excellent model to decipher the mechanisms controlling the penicillin biosynthetic pathway. In this work, we have characterized a novel transcription factor PcRFX1, which is an ortholog of the Acremonium chrysogenum CPCR1 and Penicillium marneffei RfxA regulatory proteins. PcRFX1 DNA binding sequences were found in the promoter region of the pcbAB, pcbC and penDE genes. We show in this article that these motifs control the expression of the β-galactosidase lacZ reporter gene, indicating that they may direct the PcRFX1-mediated regulation of the penicillin biosynthetic genes. By means of Pcrfx1 gene knock-down and overexpression techniques we confirmed that PcRFX1 controls penicillin biosynthesis through the regulation of the pcbAB, pcbC and penDE transcription. Morphology and development seemed not to be controlled by this transcription factor under the conditions studied and only sporulation was slightly reduced after the silencing of the Pcrfx1 gene. A genome-wide analysis of processes putatively regulated by this transcription factor was carried out in P. chrysogenum. Results suggested that PcRFX1, in addition to regulate penicillin biosynthesis, is also involved in the control of several pathways of primary metabolism.

GATA6 reporter gene reveals myocardial phenotypic heterogeneity that is related to variations in gap junction coupling

This study examined transgenic mice whose expression of a β-galactosidase (lacZ) reporter is driven by a GATA6 gene enhancer. Previous investigations established that transcription of the transgene was associated with precardiac mesoderm and primary heart tube myocardium, which decreased progressively, so that its expression was no longer observed within ventricular myocardium by midgestation. Expression of this reporter in the adult was investigated for insights into myocyte homeostasis and cardiovascular biology. Morphometric analysis determined that <1% of myocytes, often found in small clusters, express this GATA6-associated reporter in the adult heart. LacZ expression was also found in the ascending aorta. Myocardial expression of the transgene was not associated with a proliferative phenotype or new myocyte formation, as lacZ-positive myocytes neither labeled with cell division markers nor following 5-bromodeoxyuridine pulse-chase experimentation. Despite exhibiting normal adherens junctions, these myocytes appeared to exhibit decreased connexin 43 gap junctions. Treatment with the gap junctional blocker heptanol both in vivo and in culture elevated myocardial β-galactosidase activity, suggesting that deficient gap junctional communication underlies expression of the transgenic reporter. LacZ expression within the myocardium was also enhanced in response to cryoinjury and isoproterenol-induced hypertrophy. These results reveal a previously uncharacterized phenotypic heterogeneity in the myocardium and suggest that decreased gap junctional coupling leads to induction of a signaling pathway that utilizes a unique GATA6 enhancer. Upregulation of lacZ reporter gene expression following cardiac injury indicates this transgenic mouse may serve as a model for examining the transition of the heart from healthy to pathological states.

Cellular Aspects of the Regulation of the Transcriptional Activity of Nuclear Factor Kappa B (NF-κB) in Sensory Neurons in Vitro

Nuclear factor kappa B regulates the expression of a multitude of anti/proapoptotic genes, in nerve cells among others. Intranuclear acetylation processes play an important role in the mechanism regulating the activity of NF-κB. The aim of the present work was to identify the transcriptional activity of NF-κB in sensory neurons in vitro and to explain the possible regulation of NF-κB activity by modification of acetylation processes using a transgenic strain of reporter mice in which NF-κB activation leads to expression of the LacZ reporter gene. Expression of the reporter gene was found not to occur in transgenic neurons cultured either in normal growth medium or after stimulation with TNF-α, though it was induced in most neurons by in vitro exposure to the histone deacetylase inhibitor trichostatin (TSA). Thus, the transcriptional activity of NF-κB is restricted to sensory neurons be a repression mechanism mediated by histone deacetylases.

Abnormal Dosage Compensation of Reporter Genes Driven by the Drosophila Glass Multiple Reporter (GMR) Enhancer-Promoter

In Drosophila melanogaster the male specific lethal (MSL) complex is required for upregulation of expression of most X-linked genes in males, thereby achieving X chromosome dosage compensation. The MSL complex is highly enriched across most active X-linked genes with a bias towards the 3′ end. Previous studies have shown that gene transcription facilitates MSL complex binding but the type of promoter did not appear to be important. We have made the surprising observation that genes driven by the glass multiple reporter (GMR) enhancer-promoter are not dosage compensated at X-linked sites. The GMR promoter is active in all cells in, and posterior to, the morphogenetic furrow of the developing eye disc. Using phiC31 integrase-mediated targeted integration, we measured expression of lacZ reporter genes driven by either the GMR or armadillo (arm) promoters at each of three X-linked sites. At all sites, the arm-lacZ reporter gene was dosage compensated but GMR-lacZ was not. We have investigated why GMR-driven genes are not dosage compensated. Earlier or constitutive expression of GMR-lacZ did not affect the level of compensation. Neither did proximity to a strong MSL binding site. However, replacement of the hsp70 minimal promoter with a minimal promoter from the X-linked 6-Phosphogluconate dehydrogenase gene did restore partial dosage compensation. Similarly, insertion of binding sites for the GAGA and DREF factors upstream of the GMR promoter led to significantly higher lacZ expression in males than females. GAGA and DREF have been implicated to play a role in dosage compensation. We conclude that the gene promoter can affect MSL complex-mediated upregulation and dosage compensation. Further, it appears that the nature of the basal promoter and the presence of binding sites for specific factors influence the ability of a gene promoter to respond to the MSL complex.

Endocytosis of EphA receptors is essential for the proper development of the retinocollicular topographic map

Endocytosis of Eph-ephrin complexes may be an important mechanism for converting cell-cell adhesion to a repulsive interaction. Here, we show that an endocytosis-defective EphA8 mutant forms a complex with EphAs and blocks their endocytosis in cultured cells. Further, we used bacterial artificial chromosome transgenic (Tg) mice to recapitulate the anterior>posterior gradient of EphA in the superior colliculus (SC). In mice expressing the endocytosis-defective EphA8 mutant, the nasal axons were aberrantly shifted to the anterior SC. In contrast, in Tg mice expressing wild-type EphA8, the nasal axons were shifted to the posterior SC, as predicted for the enhanced repellent effect of ephrinA reverse signalling. Importantly, Rac signalling was shown to be essential for EphA-ephrinA internalization and the subsequent nasal axonal repulsion in the SC. These results indicate that endocytosis of the Eph-ephrin complex is a key mechanism by which axonal repulsion is generated for proper guidance and topographic mapping.

Transcriptional Activity of Nuclear Factor Kappa B (NF-κB) in Post-Traumatic Sensory Neurons (a histochemical study)

Nuclear factor kappa B (NF-κB) is a transcription factor that regulates the expression of a large set of genes involved in immune and inflammatory processes. It has been suggested that the release of specific cytokines following nerve damage has a stimulatory action, activating NF-κB in neurons in the spinal ganglia (SG), which may have protective influences on sensory neurons. However, the complexity of this factor has led to contradictory conclusions on the role of NF-κB in damaged SG neurons. The aim of the present work was to determine whether NF-κB is activated in sensory neurons in mature SG in conditions of peripheral nerve damage using transgenic reporter mice in which activation of NF-κB leads to expression of the lac-z reporter gene. These experiments showed that expression of β-galactosidase (β-gal) did not occur either in damaged SG neurons or in neurons on the contralateral side. However, powerful β-gal expression was seen in damaged muscle tissue. This may reflect the regressive influence of additional signal molecules on NF-κB activity in sensory neurons.

DREB1C from Medicago truncatula enhances freezing tolerance in transgenic M. truncatula and China Rose (Rosa chinensis Jacq.)

We isolated a DREB orthologue, MtDREB1C, from Medicago truncatula. Its deduced protein contains an AP2 domain of 57 amino acids. Yeast one-hybrid assay revealed that MtDREB1C specifically bound to the dehydration-responsive element (DRE) and activated the expression of HIS3 and LacZ reporter genes. In a transcriptional activation assay, coexpression of the MtDREB1C cDNA resulted in much higher (21.2 times) transactivation of the LacZ reporter gene than experiments performed without MtDREB1C. Transformation of Medicago revealed that overexpression of MtDREB1C suppressed shoot growth, and enhanced the freezing tolerance of M. truncatula. The MtDREB1C gene was transformed into China Rose (Rosa chinensis Jacq.) driven by Arabidopsisrd29A promoter. Southern-blot analysis showed that the target gene was integrated into the genome of a surviving transgenic rose plant. Northern-blot analysis illustrated that robust expression of MtDREB1C was only activated under stress conditions, and the expressed MtDREB1C mRNA reached maximum accumulation 10 h following freezing treatment. The performance of the transgenic line under freezing stress was superior to untransformed controls. This transgenic plant continued to grow, flowered under unstressed conditions, and was phenotypically normal. These facts indicate that the MtDREB1C gene, isolated from Medicago truncatula and driven by the Arabidopsis rd29A promoter, enhanced freezing tolerance in transgenic China Rose significantly without any obvious morphological or developmental abnormality.

231 SELECTION OF UBIQUITOUS AND ENDOTHELIUM-SPECIFIC PROMOTERS FOR EXPRESSING TRANSGENES IN PIG

Several promoters of different origins have been used to drive expression of transgenes in pig with variable results. However, there has not been a systemic search to identify the most reliable, ubiquitous, and tissue-specific promoters, such as endothelium-specific promoters, to drive expression of transgenes with high predictability. In this study we tested 9 ubiquitous mammalian promoters, generated by assembling enhancers, minimal promoters, and 5′untranslated region of different origins (Table 1) and 5 endothelial-specific promoters: hFlt-1 (fms-like tyrosine kinase), hICAM-2 (intercellular adhesion molecule-2), andhEndoglin promoters from human; mouse Tie-2 promoter (Schlaeger et al. 1997 PNAS 94, 3058-3063); and pig ICAM-2 promoter (Godwin et al. 2006 Xenotransplantation 13, 514-521). These promoters drive the expression of either GFP or LacZ reporter genes and were electroporated into 9 different pig cell types: endothelium-derived 2A2 and PEDSV15 cells (Seebach et al. 2001 Xenotransplantation 8, 48-61), nasal fibroblasts, kidney epithelial cells, and lung macrophages (ATCC, CRL-2528, 2842, and 2843), pig fetal fibroblasts, liver stem cells, fat-derived stem cells, and immortalized liver endothelial cells (generated in the lab). The expression was graded as week, moderate, or strong depending on the brightness of GFP signal or intensity of LacZ staining. Out of 9 ubiquitous promoters, 7 showed either low expression in all cell types or different levels of expression in some of the cell types. However, 2 promoters, hCMV-hCMV-HTLV and SV40-hCMV-HTLV, showed strong and stable expression across all 9 cell types. Among endothelial-specific promoters, pig ICAM-2 promoter showed moderate and specific expression in all 3 endothelial cell types (strong expression with the enhancer) and no expression in non-endothelial cells. Human EC-specific promoter hFlt-1 showed moderate expression in all 3 endothelial cell types, as well as in some of the non-endothelial cells such as liver stem cells, fat-derived stem cells, and lung macrophages. The other endothelial-specific human and mouse promoters did not show any expression in both endothelial and non-endothelial cells. In summary, we identified hCMV-hCMV-HTLV and SV40-hCMV-HTLV as 2 ubiquitous promoters and pICAM2 as an endothelial-specific promoter for expression of transgenes in pig. In the future, we will test these 3 promoters for in vivo expression studies. Table 1.Ubiquitous promoter characteristics

Mosaic expression of LacZ reporter gene controlled by 5′-regulatory sequences of alpha-S 1 -Casein gene in transgenic mice

Mosaic expression of LacZ reporter gene controlled by 5′-regulatory sequences of alpha-S 1 -Casein gene in transgenic mice