Encoding Reporter Genes Research Articles

Switchable and orthogonal gene expression control inside artificial cells by synthetic riboswitches.

Here we report two novel synthetic riboswitches that respond to ASP2905 and theophylline and function in reconstituted cell-free protein synthesis (CFPS) system. We encapsulated the CFPS system as well as DNA-templated encoding reporter genes regulated by these orthogonal riboswitches inside liposomes, and achieved switchable and orthogonal control over gene expression by external stimulation with the cognate ligands.

Host range, transmissibility and antigenicity of a pangolin coronavirus.

The pathogenic and cross-species transmission potential of SARS-CoV-2-related coronaviruses (CoVs) remain poorly characterized. Here we recovered a wild-type pangolin (Pg) CoV GD strain including derivatives encoding reporter genes using reverse genetics. In primary human cells, PgCoV replicated efficiently but with reduced fitness and showed less efficient transmission via airborne route compared with SARS-CoV-2 in hamsters. PgCoV was potently inhibited by US Food and Drug Administration approved drugs, and neutralized by COVID-19 patient sera and SARS-CoV-2 therapeutic antibodies in vitro. A pan-Sarbecovirus antibody and SARS-CoV-2 S2P recombinant protein vaccine protected BALB/c mice from PgCoV infection. In K18-hACE2 mice, PgCoV infection caused severe clinical disease, but mice were protected by a SARS-CoV-2 human antibody. Efficient PgCoV replication in primary human cells and hACE2 mice, coupled with a capacity for airborne spread, highlights an emergence potential. However, low competitive fitness, pre-immune humans and the benefit of COVID-19 countermeasures should impede its ability to spread globally in human populations.

Development of a Mouse Model of Hematopoietic Loss of Y Chromosome.

This protocol describes the generation of chimeric mice in which the Y chromosome is deleted from a proportion of blood cells. This model recapitulates the phenomenon of hematopoietic mosaic loss of Y chromosome (mLOY), which is frequently observed in the blood of aged men. To construct mice with hematopoietic Y chromosome loss, lineage-negative cells are isolated from the bone marrow of ROSA26-Cas9 knock-in mice. These cells are transduced with a lentivirus vector encoding a guide RNA (gRNA) that targets multiple repeats of the Y chromosome centromere, effectively removing the Y chromosome. These cells are then transplanted into lethally irradiated wildtype C57BL6 mice. Control gRNAs are designed to target either no specific region or the fourth intron of Actin gene. Transduced cells are tracked by measuring the fraction of blood cells expressing the virally encoded reporter gene tRFP. This model represents a clinically relevant model of hematopoietic mosaic loss of Y chromosome, which can be used to study the impact of mLOY on various age-related diseases. Graphical overview.

CRISPR-Cas9-mediated Precise Knock-in Edits in Zebrafish Hearts.

Clustered regularly interspaced short palindromic repeats (CRISPR) in animal models enable precise genetic manipulation for the study of physiological phenomena. Zebrafish have been used as an effective genetic model to study numerous questions related to heritable disease, development, and toxicology at the whole-organ and -organism level. Due to the well-annotated and mapped zebrafish genome, numerous tools for gene editing have been developed. However, the efficacy of generating and ease of detecting precise knock-in edits using CRISPR is a limiting factor. Described here is a CRISPR-Cas9-based knock-in approach with the simple detection of precise edits in a gene responsible for cardiac repolarization and associated with the electrical disorder, Long QT Syndrome (LQTS). This two-single-guide RNA (sgRNA) approach excises and replaces the target sequence and links a genetically encoded reporter gene. The utility of this approach is demonstrated by describing non-invasive phenotypic measurements of cardiac electrical function in wild-type and gene-edited zebrafish larvae. This approach enables the efficient study of disease-associated variants in a whole organism. Furthermore, this strategy offers possibilities for the insertion of exogenous sequences of choice, such as reporter genes, orthologs, or gene editors.

Advances in Gene Delivery Methods to Label and Modulate Activity of Upper Motor Neurons: Implications for Amyotrophic Lateral Sclerosis.

The selective degeneration of both upper motor neurons (UMNs) and lower motor neurons (LMNs) is the pathological hallmark of amyotrophic lateral sclerosis (ALS). Unlike the simple organisation of LMNs in the brainstem and spinal cord, UMNs are embedded in the complex cytoarchitecture of the primary motor cortex, which complicates their identification. UMNs therefore remain a challenging neuronal population to study in ALS research, particularly in the early pre-symptomatic stages of animal models. A better understanding of the mechanisms that lead to selective UMN degeneration requires unequivocal visualization and cellular identification of vulnerable UMNs within the heterogeneous cortical neuronal population and circuitry. Here, we review recent novel gene delivery methods developed to cellularly identify vulnerable UMNs and modulate their activity in various mouse models. A critical overview of retrograde tracers, viral vectors encoding reporter genes and transgenic reporter mice used to visualize UMNs in mouse models of ALS is provided. Functional targeting of UMNs in vivo with the advent of optogenetic and chemogenetic technology is also discussed. These exciting gene delivery techniques will facilitate improved anatomical mapping, cell-specific gene expression profiling and targeted manipulation of UMN activity in mice. These advancements in the field pave the way for future work to uncover the precise role of UMNs in ALS and improve future therapeutic targeting of UMNs.

Targeted Gene Integration into Nuclear Genome of Microalgae Using Cre/loxPRecombination System

Genetically modified microalgae have been expected to be a useful tool for bioenergy and recombinant protein production. However, random integration of transgene in the microalgae nuclear genome is susceptible to gene silencing of heterologous gene expression. Here, we attempted to perform targeted gene integration into a pre-determined nuclear genomic site ofChlamydomonas reinhardtiiusing Cre/loxPrecombination system for stable transgene expression. We constructed an expression vector plasmid encoding reporter genes (zeocin resistant gene and green fluorescent protein gene;Zeo-2A-GFP) and mutatedloxPto generate founder cells. A donor vector encodingIFNα-4and paromomycin resistant genes flanked by corresponding mutatedloxPs was constructed and introduced into founder cells together with a Cre expression vector. The optimal ratio of donor vector to Cre expression vector was determined by counting the number of paromomycin resistant colonies. For the established clones, the targeted integration was confirmed by genomic PCR using various specific primer sets. Target genes in the donor vector could be integrated into the expected genomic site ofC. reinhardtiiusing Cre/loxPsystem. RT-PCR revealed thatIFNα-4was expressed in five independent transgenic cell lines tested. This result suggests that Cre-based cell engineering is a promising approach to generate smart microalgae expressing foreign genes.

Rational design of genetically encoded reporter genes for optical imaging of apoptosis.

Apoptosis is a process in which cells are genetically regulated to cause a series of changes in morphology and metabolic activity, which ultimately lead to cell death. Apoptosis plays a vital role in the entire life cycle of an organism. Too much or too little apoptosis can cause a variety of diseases. Therefore, efficient and convenient methods for detecting apoptosis are necessary for clinical treatment and drug development. Traditional methods for detecting apoptosis may cause damage to the body during sample collection, such as for flow cytometry analysis. So it is necessary to monitor apoptosis without invasion in vivo. Optical imaging technique provides a more sensitive and economical way for apoptosis visualization. A subset of engineered reporter genes based on fluorescent proteins or luciferases are currently developed to monitor the dynamic changes in apoptotic markers, such as activation of caspases and exposure of phosphatidylserine on the surface of dying cells. These reporters detect apoptosis when cells have not undergone significant morphological changes, providing conditions for early diagnosis of tumors. In addition, these reporters show considerable value in high-throughput screening of apoptosis-related drugs and evaluation of their efficacy in treating tumors. In this review, we will discuss the recent research progress in the optical imaging of apoptosis based on the genetically encoded reporter genes.

Overexpression of T-bet, GATA-3 and TGF-ß Induces IFN-γ, IL-4/13A, and IL-17A Expression in Atlantic Salmon.

The overexpression of GATA-3, T-bet and TGF-ß may theoretically induce IL-4/A, IFN-γ and IL-17A expression, respectively. Whether this also applies to fish is not yet known. The plasmid vectors encoding reporter gene (RFP)-tagged T-bet, GATA-3 and TGF-ß were used as overexpression tools, transfected into cells or injected intramuscularly to monitor the expression of IFN-γ, IL-4/13A and IL-17A. In addition, the fish were either experimentally challenged with Vibrio anguillarum (VA group) or Piscirickettsia salmonis (PS group). The reporter gene (RFP) inserted upstream of the GATA-3, T-bet and TGF-ß genes, was observed in muscle cell nuclei and in inflammatory cells after intramuscular (i.m.) injection. PS group: following the injection of GATA-3 and T-bet-encoding plasmids, the expression of GATA-3 and T-bet was high at the injection site. The spleen expression of IFN-γ, following the injection of a T-bet-encoding plasmid, was significantly higher on day 2. VA group: The T-bet and GATA-3-overexpressing fish expressed high T-bet and GATA-3 mRNA levels in the muscles and on day 4 post-challenge. The expression of TGF-ß in the muscles of fish injected with TGF-ß-encoding plasmids was significantly higher on days 7 (8 days pre-challenge) and 19 (4 days after challenge). The protective effects of the overexpression of T-bet, GATA-3 and TGF-ß on both bacterial infections were negligible.

New insights on the role of ROS in the mechanisms of sonoporation-mediated gene delivery.

Reactive oxygen species (ROS) are hypothesized to play a role in the sonoporation mechanisms. Nevertheless, the acoustical phenomenon behind the ROS production as well as the exact mechanisms of ROS action involved in the increased cell membrane permeability are still not fully understood. Therefore, we investigated the key processes occurring at the molecular level in and around microbubbles subjected to ultrasound using computational chemistry methods. To confirm the molecular simulation predictions, we measured the ROS production by exposing SonoVue® microbubbles (MBs) to ultrasound using biological assays. To investigate the role of ROS in cell membrane permeabilization, cells were subjected to ultrasound in presence of MBs and plasmid encoding reporter gene, and the transfection level was assessed using flow cytometry. The molecular simulations showed that under sonoporation conditions, ROS can form inside the MBs. These radicals could easily diffuse through the MB shell toward the surrounding aqueous phase and participate in the permeabilization of nearby cell membranes. Experimental data confirmed that MBs favor spontaneous formation of a host of free radicals where HO was the main ROS species after US exposure. The presence of ROS scavengers/inhibitors during the sonoporation process decreased both the production of ROS and the subsequent transfection level without significant loss of cell viability. In conclusion, the exposure of MBs to ultrasound might be the origin of chemical effects, which play a role in the cell membrane permeabilization and in the in vitro gene delivery when generated in its proximity.

Cationic Derivatives of Polyisoprenoids and Commercial Lipid‐Based DNA Carriers for Transfection in vivo in Spontaneously Hypertensive Rats

Development of efficient vectors for transfection constitutes a major challenge in modern gene therapy. Viral vectors are considered more effective, but plagued by safety concerns. Research shows that derivatives of polyisoprenoid alcohols (trimethylpolyprenylammonium iodides, called PTAI), may serve as lipofectants. In previous research PTAI were proved to be effective as DNA carriers in vitro and in vivo as carriers of DNA vaccines against influenza virus.In the present study the effectiveness of two compositions (PTAI‐6–8 and PTAI‐10–14, containing a mixture of polyprenols with chain lengths from 6 to 8 subunits and from 10 to 14 subunits, respectively and helper lipids: DOPE (1,2‐dioleoyl‐sn‐glycero‐3‐phosphatidylethanolamine) and DC‐cholesterol (3β‐[N‐(N′,N′‐dimethylaminoethane)‐carbamoyl]cholesterol hydrochloride) was investigated for DNA delivery into rats' cells and compared to the commercially available reagents for transfection: in vivo‐jetPEI® (Polyplus‐transfection) and Avalanche®‐in vivo Transfection Reagent (EZ Biosystems LLC). Selected mixtures were used to enhance the expression of vascular endothelial growth factor type A (VEGF‐A) in spontaneously hypertensive rats (SHR). VEGF deficiency is considered one of the factors in the development of hypertension and related kidney diseases.In preliminary experiments, the mixtures containing lipid‐carriers and plasmid DNA (encoding reporter gene and VEGF‐A) were administered intravenously and observed for 24, 48 or 72 h, to establish the optimal conditions for transfection. Subsequently rats were anesthetized, perfused with PBS, and tissues (liver, spleen, kidney, lung) were collected for analysis. Transfection efficiency was evaluated by flow cytometry (BD FACSCanto™ II system) in single cell suspensions prepared from collected tissues.In almost all tested cell suspensions the level of fluorescence of reporter gene (eGFP or mCherry) was undetectable. Only in the liver higher expression of mCherry was observed with commercial reagents (the % of mCherry positive liver cells with Avalanche® was 42 versus 12% in control group receiving 5% glucose; p<0.05). Due to low efficiency of intravenously applied carriers, we tested if selected mixtures are able to provide stable expression of VEGF‐A gene after direct administration to the kidney. SHR were implanted with osmotic pumps (model 2001, ALZET®) connected to catheters allowing the infusion into the renal medulla.The fluorescence of mCherry in the kidneys was undetectable and there were no changes in tissue, plasma and urine levels of VEGF‐A (ELISA). Unfortunately we were not able to prove the usefulness of PTAI for transfection, however all selected mixtures, including commercially available reagents failed to deliver DNA to renal cellsSupport or Funding InformationNCN 2015/17/N/NZ4/02831This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A novel approach for assessment of prostate cancer aggressiveness using survivin-driven tumour-activatable minicircles

Early and accurate detection of cancer is essential to optimising patient outcomes. Of particular importance to prostate cancer is the ability to determine the aggressiveness of a primary tumour, which allows for effective management of patient care. In this work, we propose using gene vectors called tumour-activatable minicircles which deliver an exogenously encoded reporter gene into cancer cells, forcing them to produce a unique and sensitive biomarker. These minicircles express a blood reporter protein called secreted embryonic alkaline phosphatase mediated by the tumour-specific survivin promoter, which exhibits activity graded to prostate cancer aggressiveness. Together, these components underlie a detection system where levels of blood reporter are indicative of not only the presence, but also the metastatic potential of a tumour. Our goal was to assess the ability of tumour-activatable minicircles to detect and characterise primary prostate lesions. Our minicircles produced reporter levels related to survivin expression across a range of prostate cancer cell lines. When survivin-driven minicircles were administered intratumourally into mice, reporter levels in blood samples were significantly higher (p < 0.05) in mice carrying prostate tumours of high versus low-aggressiveness. Continued development of this gene-based system could provide clinicians with a powerful tool to evaluate prostate cancer aggressiveness using a sensitive and affordable blood assay.

MRNA transfection by a Xentry-protamine cell-penetrating peptide is enhanced by TLR antagonist E6446.

Messenger RNA (mRNA) transfection is a developing field that has applications in research and gene therapy. Potentially, mRNA transfection can be mediated efficiently by cell-penetrating peptides (CPPs) as they may be modified to target specific tissues. However, whilst CPPs are well-documented to transfect oligonucleotides and plasmids, mRNA transfection by CPPs has barely been explored. Here we report that peptides, including a truncated form of protamine and the same peptide fused to the CPP Xentry (Xentry-protamine; XP), can transfect mRNAs encoding reporter genes into human cells. Further, this transfection is enhanced by the anti-malarial chloroquine (CQ) and the toll-like receptor antagonist E6446 (6-[3-(pyrrolidin-1-yl)propoxy)-2-(4-(3-(pyrrolidin-1-yl)propoxy)phenyl]benzo[d]oxazole), with E6446 being >5-fold more potent than CQ at enhancing this transfection. Finally, E6446 facilitated the transfection by XP of mRNA encoding the cystic fibrosis transmembrane regulator, the protein mutated in cystic fibrosis. As such, these findings introduce E6446 as a novel transfection enhancer and may be of practical relevance to researchers seeking to improve the mRNA transfection efficiency of their preferred CPP.

Monitoring ecdysteroid activities using genetically encoded reporter gene in Daphnia magna

Ecdysteroid is an important hormone that regulates growth, reproduction, and embryogenesis in arthropods. However, little is known about its role and action mechanism in crustaceans, despite their pivotal role in aquatic ecosystem. Daphnia magna, a freshwater crustacean, is used as a classic model organism in ecology and ecotoxicology. Its ecdysteroid activity has been partially characterized previously. However, the spatio-temporal behavior of ecdysteroid, especially during early embryogenesis, when it is thought to have pivotal roles, is still unclear. Thus, we proposed a genetic modification approach by integrating a reporter gene exhibiting ecdysteroid activity in vivo.We used the clustered regularly interspaced palindromic repeats (CRISPR) genome editing technique, followed by non-homologous end-joining (NHEJ) pathway as the transgenesis method to generate the ecdysteroid reporter transgenic Daphnia. One transgenic Daphnia containing one copy of the ecdysone response element (EcRE)-controlled reporter gene mCherry was successfully obtained and was designated EcRE-mCh.The expression of mCherry was observed during early embryogenesis starting from 12 h after ovulation (hao). The time-lapse imaging during 12–24 hao showed the growing expression of mCherry signal originating from the posterior section of embryo and then migrating toward the anterior section. From 18 hao, the signal was detected around the developing thoracic appendages and localized between the first to third thoracic segments. The establishment of this EcRE-mCh line and its ability to exhibit ecdysteroid activity spatio-temporally might serve as convenient tool to elucidate the roles of ecdysteroid during the early stage of animal development. Moreover, the expression of mCherry in response to the presence of ecdysteroid in water suggests that EcRE-mCh could be used for monitoring ecdysteroid activities in environmental water.

Genomic integration and ligand-dependent activation of the human estrogen receptor α in the crustacean Daphnia magna.

The freshwater crustacean Daphnia have a long history in water quality assessments and now lend themselves to detection of targeted chemicals using genetically encoded reporter gene due to recent progress in the development of genome editing tools. By introducing human genes into Daphnia, we may be able to detect chemicals that affect the human system, or even apply it to screening potentially useful chemicals. Here, we aimed to develop a transgenic line of Daphnia magna that contains the human estrogen receptor alpha (hERα) and shows a fluorescence response to exposure of estrogens. We designed plasmids to express hERα in Daphnia (EF1α1:esr1) and to report estrogenic activity via red fluorescence (ERE:mcherry) under the control of estrogen response element (ERE). After confirmation of functionality of the plasmids by microinjection into embryos, the two plasmids were joined, a TALE site was added and integrated into the D. magna genome using TALEN. When the resulting transgenic Daphnia named the ES line was exposed to Diethylstilbestrol (DES) or 17β-Estradiol (E2), the ES line could reliably expressed red fluorescence derived from mCherry in a ligand-dependent manner, indicating that an estrogen-responsive line of D. magna was established. This is the first time a human gene was expressed in Daphnia, showcasing potential for further research.

Real time analysis of viral infection: contribution of the zebrafish model to visualization and characterization of host/pathogen interactions.

Real time analysis of viral infection is now feasible with the emergence of viral mutants encoding reporter genes (such as fluorescent proteins or Luciferase). The development of reverse genetic approaches in the virology field has contributed significantly to improve our knowledge on viral dissemination in mammals using intravital imaging technologies. In this context, zebra fish recently appeared as a promizing tool to study infectiology and viruses responsible for human diseases such as Herpes, Flu, hepatitis and chikungunya infections. This small vertebrate is optically transparent during the early stages of development, and has been particularly useful to study the tropism of fluorescent viruses, spreading mechanisms as well as viral persistence at cellular resolution. The size of this small organism is compatible with imaging of the whole larva using a dissecting microscope. Genome editing technology has contributed important tools that are now being exploited for the characterization of the host pathogen interaction in vivo, mainly focused on the conserved features of antiviral innate immunity.

Role of Endonuclease G in Exogenous DNA Stability in HeLa Cells.

Endonuclease G (EndoG) is a well-conserved mitochondrial-nuclear nuclease with dual lethal and vital roles in the cell. The aim of our study was to examine whether EndoG exerts its nuclease activity on exogenous DNA substrates such as plasmid DNA (pDNA), considering their importance in gene therapy applications. The effects of EndoG knockdown on pDNA stability and levels of encoded reporter gene expression were evaluated in the cervical carcinoma HeLa cells. Transfection of pDNA vectors encoding short-hairpin RNAs (shRNAs) reduced levels of EndoG mRNA in HeLa cells. In physiological circumstances, EndoG knockdown did not have an effect on the stability of pDNA or the levels of encoded transgene expression as measured over a four-day time course. However, when endogenous expression of EndoG was induced by an extrinsic stimulus, targeting of EndoG by shRNA improved the perceived stability and transgene expression of pDNA vectors. Therefore, EndoG is not a mediator of exogenous DNA clearance, but in non-physiological circumstances, it may nonspecifically cleave intracellular DNA regardless of its origin. These findings make it unlikely that targeting of EndoG is a viable strategy for improving the duration and level of transgene expression from nonviral DNA vectors in gene therapy efforts.

Non-Viral, Lipid-Mediated DNA and mRNA Gene Therapy of the Central Nervous System (CNS): Chemical-Based Transfection.

Appropriate gene delivery systems are essential for successful gene therapy in clinical medicine. Cationic lipid-mediated delivery is an alternative to viral vector-mediated gene delivery. Lipid-mediated delivery of DNA or mRNA is usually more rapid than viral-mediated delivery, offers a larger payload, and has a nearly zero risk of incorporation. Lipid-mediated delivery of DNA or RNA is therefore preferable to viral DNA delivery in those clinical applications that do not require long-term expression for chronic conditions. Delivery of RNA may be preferable to non-viral DNA delivery in some clinical applications, because transit across the nuclear membrane is not necessary and onset of expression with RNA is therefore even faster than with DNA, although both are faster than most viral vectors. Here, we describe techniques for cationic lipid-mediated delivery of nucleic acids encoding reporter genes in a variety of cell lines. We describe optimized formulations and transfection procedures that we previously assessed by bioluminescence and flow cytometry. RNA transfection demonstrates increased efficiency relative to DNA transfection in non-dividing cells. Delivery of mRNA results in onset of expression within 1 h after transfection and a peak in expression 5-7 h after transfection. Duration of expression in eukaryotic cells after mRNA transcript delivery depends on multiple factors, including transcript stability, protein turnover, and cell type. Delivery of DNA results in onset of expression within 5 h after transfection, a peak in expression 24-48 h after transfection, and a return to baseline that can be as long as several weeks after transfection. In vitro results are consistent with our in vivo delivery results, techniques for which are described as well. RNA delivery is suitable for short-term transient gene expression due to its rapid onset, short duration of expression and greater efficiency, particularly in non-dividing cells, while the longer duration and the higher mean levels of expression per cell that are ultimately obtained following DNA delivery confirm a continuing role for DNA gene delivery in clinical applications that require longer term transient gene expression.

Noninvasive Imaging Reveals Stable Transgene Expression in Mouse Airways After Delivery of a Nonintegrating Recombinant Adeno-Associated Viral Vector.

Gene therapy holds promise to cure a wide range of genetic and acquired diseases. Recent successes in recombinant adeno-associated viral vector (rAAV)-based gene therapy in the clinic for hereditary disorders such as Leber's congenital amaurosis and hemophilia B encouraged us to reexplore an rAAV approach for pulmonary gene transfer. Only limited clinical successes have been achieved for airway gene transfer so far, underscoring the need for further preclinical development of rAAV-based gene therapy for pulmonary disorders. We sought to determine the preclinical potential of an airway-tropic serotype, rAAV2/5, encoding reporter genes when delivered to mouse airways. Although several groups have assessed the stability of gene transfer using a nonintegrating rAAV in mouse airways, long-term stability for more than a year has not been reported. Additionally, an extensive quantitative analysis of the specific cell types targeted by rAAV2/5 using cell-specific markers is lacking. We obtained sustained gene expression in upper and lower airways up to 15 months after vector administration, a substantial proportion of the lifespan of a laboratory mouse. In addition, we demonstrated that readministration of rAAV2/5 to the airways is feasible and increases gene expression 14 months after primary vector administration, despite the presence of circulating neutralizing antibodies. Finally, identification of transduced cell types revealed different subpopulations being targeted by rAAV2/5, with 64% of β-galactosidase-positive cells being ciliated cells, 34% club cells in the conducting airways, and 75% alveolar type II cells in the alveoli at 1 month postinjection. This underscores the therapeutic potential of a nonintegrating rAAV vector to develop a gene therapeutic drug for a variety of pulmonary disorders, such as cystic fibrosis, primary ciliary dyskinesia, and surfactant deficiencies.

In vivo microinjection and electroporation of mouse testis.

This video and article contribution gives a comprehensive description of microinjection and electroporation of mouse testis in vivo. This particular transfection technique for testicular mouse cells allows the study of unique processes in spermatogenesis. The following protocol focuses on transfection of testicular mouse cells with plasmid constructs. Specifically, we used the reporter vector pEGFP-C1, which expresses enhanced green fluorescent protein (eGFP) and also the pDsRed2-N1 vector expressing red fluorescent protein (DsRed2). Both encoded reporter genes were under the control of the human cytomegalovirus immediate-early promoter (CMV). For performing gene transfer into mouse testes, the reporter plasmid constructs are injected into testes of living mice. To that end, the testis of an anaesthetized animal is exposed and the site of microinjection is prepared. Our preferred place of injection is the efferent duct, with the ultimately connected rete testis as the anatomical transport route of the spermatozoa between the testis and the epididymis. In this way, the filling of the seminiferous tubules after microinjection is excellently managed and controlled due to the use of stained DNA solutions. After observing a sufficient filling of the testis by its colored tubule structure, the organ is electroporated. This enables the transfer of the DNA solution into the testicular cells. Following 3 days of incubation, the testis is removed and investigated under the microscope for green or red fluorescence, illustrating transfection success. Generally, this protocol can be employed for delivering DNA- or RNA- constructs into living mouse testis in order to (over)express or knock down genes, facilitating in vivo gene function analysis. Furthermore, it is suitable for studying reporter constructs or putative gene regulatory elements. Thus, the main advantages of the electroporation technique are fast performance in combination with low effort as well as the moderate technical equipment and skills required compared to alternative techniques.

Distinct HIV-1 entry phenotypes are associated with transmission, subtype specificity, and resistance to broadly neutralizing antibodies.

BackgroundThe efficiency of CD4/CCR5 mediated HIV-1 entry has important implications for pathogenesis and transmission. The HIV-1 receptor affinity profiling (Affinofile) system analyzes and quantifies the infectivity of HIV-1 envelopes (Envs) across a spectrum of CD4/CCR5 expression levels and distills these data into a set of Affinofile metrics. The Affinofile system has shed light on how differential CD4/CCR5 usage efficiencies contributes to an array of Env phenotypes associated with cellular tropism, viral pathogenesis, and CCR5 inhibitor resistance. To facilitate more rapid, convenient, and robust analysis of HIV-1 entry phenotypes, we engineered a reporter Affinofile system containing a Tat- and Rev-dependent Gaussia luciferase-eGFP-Reporter (GGR) that is compatible with the use of pseudotyped or replication competent viruses with or without a virally encoded reporter gene. This GGR Affinofile system enabled a higher throughput characterization of CD4/CCR5 usage efficiencies associated with differential Env phenotypes.ResultsWe first validated our GGR Affinofile system on isogenic JR-CSF Env mutants that differ in their affinity for CD4 and/or CCR5. We established that their GGR Affinofile metrics reflected their differential entry phenotypes on primary PBMCs and CD4+ T-cell subsets. We then applied GGR Affinofile profiling to reveal distinct entry phenotypes associated with transmission, subtype specificity, and resistance to broadly neutralizing antibodies (BNAbs). First, we profiled a panel of reference subtype B transmitted/founder (T/F) and chronic Envs (n = 12) by analyzing the infectivity of each Env across 25 distinct combinations of CD4/CCR5 expression levels. Affinofile metrics revealed that at low CCR5 levels, our panel of subtype B T/F Envs was more dependent on high levels of CD4 for HIV-1 entry compared to chronic Envs. Next, we analyzed a reference panel of 28 acute/early subtype A-D Envs, and noted that subtype C Envs could be distinguished from the other subtypes based on their infectivity profiles and relevant Affinofile metrics. Lastly, mutations known to confer resistance to VRC01 or PG6/PG19 BNAbs, when engineered into subtypes A-D Envs, resulted in significantly decreased CD4/CCR5 usage efficiency.ConclusionsGGR Affinofile profiling reveals pathophysiological phenotypes associated with varying HIV-1 entry efficiencies, and highlight the fitness costs associated with resistance to some broadly neutralizing antibodies.