Search

Abstract Background Transgenic (Tg) mice are widely used in biomedical research, and they are typically generated by injecting transgenic DNA cassettes into pronuclei of one-cell stage zygotes. Such animals often show unreliable expression of the transgenic DNA, one of the major reasons for which is random insertion of the transgenes. We previously developed a method called “pronuclear injection-based targeted transgenesis” (PITT), in which DNA constructs are directed to insert at pre-designated genomic loci. PITT was achieved by pre-installing so called landing pad sequences (such as heterotypic LoxP sites or attP sites) to create seed mice and then injecting Cre recombinase or PhiC31 integrase mRNAs along with a compatible donor plasmid into zygotes derived from the seed mice. PITT and its subsequent version, improved PITT (i-PITT), overcome disadvantages of conventional Tg mice such as lack of consistent and reliable expression of the cassettes among different Tg mouse lines, and the PITT approach is superior in terms of cost and labor. One of the limitations of PITT, particularly using Cre-mRNA, is that the approach cannot be used for insertion of conditional expression cassettes using Cre-LoxP site-specific recombination. This is because the LoxP sites in the donor plasmids intended for achieving conditional expression of the transgene will interfere with the PITT recombination reaction with LoxP sites in the landing pad. Results To enable the i-PITT method to insert a conditional expression cassette, we modified the approach by simultaneously using PhiC31o and FLPo mRNAs. We demonstrate the strategy by creating a model containing a conditional expression cassette at the Rosa26 locus with an efficiency of 13.7%. We also demonstrate that inclusion of FLPo mRNA excludes the insertion of vector backbones in the founder mice. Conclusions Simultaneous use of PhiC31 and FLP in i-PITT approach allows insertion of donor plasmids containing Cre-loxP-based conditional expression cassettes.

Genome engineered B-cells are being developed for chronic, systemic in vivo protein replacement therapies and for localized, tumor cell-actuated anticancer therapeutics. For continuous systemic engineered protein production, expression may be driven by constitutively active promoters. For actuated payload delivery, B-cell conditional expression could be based on transgene alternate splicing or heterologous promotors activated after engineered B-cell receptor (BCR) stimulation. This study used a bioinformatics-based approach to identify putative BCR-stimulated gene promoters. Gene expression data at four timepoints (60, 90, 210, and 390 min) following in vitro BCR stimulation using an anti-IgM antibody in B-cells from six healthy donors were analyzed using R (4.2.2). Differentially upregulated genes were stringently defined as those with adjusted p-value < 0.01 and a log2FoldChange > 1.5. The most upregulated and statistically significant genes were further analyzed to find those with the lowest unstimulated B-cell expression. Of the 46 significantly upregulated genes at 390 min post-BCR stimulation, 6 had average unstimulated expression below the median unstimulated expression at 390 min for all 54,675 gene probes. This bioinformatics-based identification of 6 relatively quiescent genes at baseline that are upregulated by BCR-stimulation (“on-switch”) provides a set of promising promotors for inclusion in future transgene designs and engineered B-cell therapeutics development.

Background: Transgenic (Tg) mice are widely used in biomedical research, and they are typically generated by injecting transgenic DNA cassettes into pronuclei of one-cell stage zygotes. Such animals often show unreliable expression of the transgenic DNA, one of the major reasons for which is random insertion of the transgenes. We previously developed a method called "pronuclear injection-based targeted transgenesis" (PITT), in which DNA constructs are directed to insert at pre-designated genomic loci. PITT was achieved by pre-installing so called landing pad sequences (such as heterotypic LoxP sites or attP sites) to create seed mice and then injecting Cre recombinase or PhiC31 integrase mRNAs along with a compatible donor plasmid into zygotes derived from the seed mice. PITT and its subsequent version, improved PITT (i-PITT), overcome disadvantages of conventional Tg mice such as lack of consistent and reliable expression of the cassettes among different Tg mouse lines, and the PITT approach is superior in terms of cost and labor. One of the limitations of PITT, particularly using Cre-mRNA, is that the approach cannot be used for insertion of conditional expression cassettes using Cre-LoxP site-specific recombination. This is because the LoxP sites in the donor plasmids intended for achieving conditional expression of the transgene will interfere with the PITT recombination reaction with LoxP sites in the landing pad. Results: To enable the i-PITT method to insert a conditional expression cassette, we modified the approach by simultaneously using PhiC31o and FLPo mRNAs. We demonstrate the strategy by creating a model containing a conditional expression cassette at the Rosa26 locus with an efficiency of 13.7%. We also demonstrate that inclusion of FLPo mRNA excludes the insertion of vector backbones in the founder mice. Conclusions: Simultaneous use of PhiC31 and FLP in i-PITT approach allows insertion of donor plasmids containing Cre-loxP-based conditional expression cassettes. [ABSTRACT FROM AUTHOR]

Genome engineered B-cells are being developed for chronic, systemic in vivo protein replacement therapies and for localized, tumor cell-actuated anticancer therapeutics. For continuous systemic engineered protein production, expression may be driven by constitutively active promoters. For actuated payload delivery, B-cell conditional expression could be based on transgene alternate splicing or heterologous promotors activated after engineered B-cell receptor (BCR) stimulation. This study used a bioinformatics-based approach to identify putative BCR-stimulated gene promoters. Gene expression data at four timepoints (60, 90, 210, and 390 min) following in vitro BCR stimulation using an anti-IgM antibody in B-cells from six healthy donors were analyzed using R (4.2.2). Differentially upregulated genes were stringently defined as those with adjusted p-value < 0.01 and a log2FoldChange > 1.5. The most upregulated and statistically significant genes were further analyzed to find those with the lowest unstimulated B-cell expression. Of the 46 significantly upregulated genes at 390 min post-BCR stimulation, 6 had average unstimulated expression below the median unstimulated expression at 390 min for all 54,675 gene probes. This bioinformatics-based identification of 6 relatively quiescent genes at baseline that are upregulated by BCR-stimulation ("on-switch") provides a set of promising promotors for inclusion in future transgene designs and engineered B-cell therapeutics development. [ABSTRACT FROM AUTHOR]

Human cytomegalovirus (HCMV) infection is associated with severe disease conditions either following congenital transmission of the virus or viral reactivation in immunosuppressed individuals. Consequently, the establishment of a protective vaccine is of high medical need. Several candidates have been tested in preclinical and clinical studies, yet no vaccine has been licensed. Subviral dense bodies (DB) are a promising vaccine candidate. We have recently provided a GMP-compliant protocol for the production of DB, based on a genetically modified version of the HCMV laboratory strain Towne, expressing the pentameric complex of envelope protein gH-gL-pUL128-131 (Towne-UL130rep). In this work, we genetically attenuated Towne-UL130rep by abrogating the expression of the tegument protein pUL25 and by fusing the destabilizing domain ddFKBP to the N-terminus of the IE1- and IE2-proteins of HCMV. The resulting strain, termed TR-VAC, produced high amounts of DB under IE1/IE2 repressive conditions and concomitant supplementation of the viral terminase inhibitor letermovir to the producer cell culture. TR-VAC DB retained the capacity to induce neutralizing antibodies. A complex pattern of host protein induction was observed by mass spectrometry following exposure of primary human monocytes with TR-VAC DB. Human monocyte-derived dendritic cells (DC) moderately increased the expression of activation markers and MHC molecules upon stimulation with TR-VAC DB. In a co-culture with autologous T cells, the TR-VAC DB-stimulated DC induced a robust HCMV-specific T cell-activation and –proliferation. Exposure of donor-derived monocytic cells to DB led to the activation of a rapid innate immune response. This comprehensive data set thus shows that TR-VAC is an optimal attenuated seed virus strain for the production of a DB vaccine to be tested in clinical studies. [ABSTRACT FROM AUTHOR]

Herpesviral nuclear egress is a fine-tuned regulatory process that defines the nucleocytoplasmic release of viral capsids. Nuclear capsids are unable to traverse via nuclear pores due to the fact of their large size; therefore, herpesviruses evolved to develop a vesicular transport pathway mediating the transition across the two leaflets of the nuclear membrane. The entire process involves a number of regulatory proteins, which support the local distortion of the nuclear envelope. In the case of the prototype species of β-Herpesvirinae, the human cytomegalovirus (HCMV), the nuclear egress complex (NEC) is determined by the core proteins pUL50 and pUL53 that oligomerize, form capsid docking lattices and mediate multicomponent assembly with NEC-associated viral and cellular proteins. The NEC-binding principle is based on the hook-into-groove interaction through an N-terminal hook-like pUL53 protrusion that embraces an α-helical pUL50 binding groove. Thus far, the function and characteristics of herpesviral core NECs have been well studied and point to the groove proteins, such as pUL50, as the multi-interacting, major determinants of NEC formation and egress. This review provides closer insight into (i) sequence and structure conservation of herpesviral core NEC proteins, (ii) experimentation on cross-viral core NEC interactions, (iii) the essential functional roles of hook and groove proteins for viral replication, (iv) an establishment of assay systems for NEC-directed antiviral research and (v) the validation of NEC as putative antiviral drug targets. Finally, this article provides new insights into the conservation, function and antiviral targeting of herpesviral core NEC proteins and, into the complex regulatory role of hook and groove proteins during the assembly, egress and maturation of infectious virus. [ABSTRACT FROM AUTHOR]

Human cytomegalovirus (HCMV) infection is associated with severe disease conditions either following congenital transmission of the virus or viral reactivation in immunosuppressed individuals. Consequently, the establishment of a protective vaccine is of high medical need. Several candidates have been tested in preclinical and clinical studies, yet no vaccine has been licensed. Subviral dense bodies (DB) are a promising vaccine candidate. We have recently provided a GMP-compliant protocol for the production of DB, based on a genetically modified version of the HCMV laboratory strain Towne, expressing the pentameric complex of envelope protein gH-gL-pUL128-131 (Towne-UL130rep). In this work, we genetically attenuated Towne-UL130rep by abrogating the expression of the tegument protein pUL25 and by fusing the destabilizing domain ddFKBP to the N-terminus of the IE1- and IE2-proteins of HCMV. The resulting strain, termed TR-VAC, produced high amounts of DB under IE1/IE2 repressive conditions and concomitant supplementation of the viral terminase inhibitor letermovir to the producer cell culture. TR-VAC DB retained the capacity to induce neutralizing antibodies. A complex pattern of host protein induction was observed by mass spectrometry following exposure of primary human monocytes with TR-VAC DB. Human monocyte-derived dendritic cells (DC) moderately increased the expression of activation markers and MHC molecules upon stimulation with TR-VAC DB. In a co-culture with autologous T cells, the TR-VAC DB-stimulated DC induced a robust HCMV-specific T cell-activation and –proliferation. Exposure of donor-derived monocytic cells to DB led to the activation of a rapid innate immune response. This comprehensive data set thus shows that TR-VAC is an optimal attenuated seed virus strain for the production of a DB vaccine to be tested in clinical studies.

Brucella, the bacterial agent of common zoonotic brucellosis, primarily infects specific animal species. The Brucella outer membrane proteins (Omps) are particularly attractive for developing vaccine and improving diagnostic tests and are associated with the virulence of smooth Brucella strains. Omp16 is a homologue to peptidoglycan-associated lipoproteins (Pals), and an omp16 mutant has not been generated in any Brucella strain until now. Very little is known about the functions and pathogenic mechanisms of Omp16 in Brucella. Here, we confirmed that Omp16 has a conserved Pal domain and is highly conserved in Brucella. We attempted to delete omp16 in Brucella suis vaccine strain 2 (B. suis S2) without success, which shows that Omp16 is vital for Brucella survival. We acquired a B. suis S2 Omp16 mutant via conditional complementation. Omp16 deficiency impaired Brucella outer membrane integrity and activity in vitro. Moreover, inactivation of Omp16 decreased bacterial intracellular survival in macrophage RAW 264.7 cells. B. suis S2 and its derivatives induced marked expression of IL-1β, IL-6, and TNF-a mRNA in Raw 264.7 cells. Whereas inactivation of Omp16 in Brucella enhanced IL-1β and IL-6 expression in Raw 264.7 cells. Altogether, these findings show that the Brucella Omp16 mutant was obtained via conditional complementation and confirmed that Omp16 can maintain outer membrane integrity and be involved in bacterial virulence in Brucella in vitro and in vivo. These results will be important in uncovering the pathogenic mechanisms of Brucella. [ABSTRACT FROM AUTHOR]

The regulation of the nucleocytoplasmic release of herpesviral capsids is defined by the process of nuclear egress. Due to their large size, nuclear capsids are unable to traverse via nuclear pores, so that herpesviruses evolved to develop a vesicular transport pathway mediating their transition through both leaflets of the nuclear membrane. This process involves regulatory proteins, which support the local distortion of the nuclear envelope. For human cytomegalovirus (HCMV), the nuclear egress complex (NEC) is determined by the pUL50-pUL53 core that initiates multicomponent assembly with NEC-associated proteins and capsids. Hereby, pUL50 serves as a multi-interacting determinant that recruits several viral and cellular factors by direct and indirect contacts. Recently, we generated an ORF-UL50-deleted recombinant HCMV in pUL50-complementing cells and obtained first indications of putative additional functions of pUL50. In this study, we produced purified ΔUL50 particles under both complementing (ΔUL50C) and non-complementing (ΔUL50N) conditions and performed a phenotypical characterization. Findings were as follows: (i) ΔUL50N particle preparations exhibited a clear replicative defect in qPCR-based infection kinetics compared to ΔUL50C particles; (ii) immuno-EM analysis of ΔUL50C did not reveal major changes in nuclear distribution of pUL53 and lamin A/C; (iii) mass spectrometry-based quantitative proteomics showed a large concordance of protein contents in the NIEP fractions of ΔUL50C and ΔUL50N particles, but virion fraction was close to the detection limit for ΔUL50N; (iv) confocal imaging of viral marker proteins of immediate early (IE) and later phases of ΔUL50N infection indicated a very low number of cells showing an onset of viral lytic protein expression; and, finally (v) quantitative measurements of encapsidated genomes provided evidence for a substantial reduction in the DNA contents in ΔUL50N compared to ΔUL50C particles. In summary, the results point to a complex and important regulatory role of the HCMV nuclear egress protein pUL50 in the maturation of infectious virus.

Background: Regeneration of fertile plants from tissue culture is a critical bottleneck in the application of new plant breeding technologies. Ectopic overexpression of morphogenic factors is a promising workaround for this hurdle. Methods: Conditional overexpression of WUS and ARF5Δ was used to study the effect of timing the overexpression of these morphogenic factors during shoot regeneration from root explants in Arabidopsis. In addition, their effect on auxin-signaling activation was examined by visualization and cytometric quantification of the DR5:GFP auxin-signaling reporter in roots and protoplasts, respectively. Results: The induced expression of both WUS and ARF5Δ led to an activation of auxin signaling in roots. Activation of auxin signaling by WUS and ARF5Δ was further quantified by transient transformation of protoplasts. Ectopic overexpression of both WUS and ARF5Δ enhanced regeneration efficiency, but only during the shoot-induction stage of regeneration and not during the callus-induction stage. Conclusions: The overexpression of WUS and ARF5Δ both lead to activation of auxin signaling. Expression during the shoot-induction stage is critical for the enhancement of shoot regeneration by WUS and ARF5Δ.