The construction of cDNA clones encoding large-size RNA molecules of biological interest, like coronavirus genomes, which are among the largest mature RNA molecules known to biology, has been hampered by the instability of those cDNAs in bacteria. Herein, we show that the application of two strategies, cloning of the cDNAs into a bacterial artificial chromosome and nuclear expression of RNAs that are typically produced within the cytoplasm, is useful for the engineering of large RNA molecules. A cDNA encoding an infectious coronavirus RNA genome has been cloned as a bacterial artificial chromosome. The rescued coronavirus conserved all of the genetic markers introduced throughout the sequence and showed a standard mRNA pattern and the antigenic characteristics expected for the synthetic virus. The cDNA was transcribed within the nucleus, and the RNA translocated to the cytoplasm. Interestingly, the recovered virus had essentially the same sequence as the original one, and no splicing was observed. The cDNA was derived from an attenuated isolate that replicates exclusively in the respiratory tract of swine. During the engineering of the infectious cDNA, the spike gene of the virus was replaced by the spike gene of an enteric isolate. The synthetic virus replicated abundantly in the enteric tract and was fully virulent, demonstrating that the tropism and virulence of the recovered coronavirus can be modified. This demonstration opens up the possibility of employing this infectious cDNA as a vector for vaccine development in human, porcine, canine, and feline species susceptible to group 1 coronaviruses.
Targeted recombination within the S (spike) gene of transmissible gastroenteritis coronavirus (TGEV) was promoted by passage of helper respiratory virus isolates in cells transfected with a TGEV-derived defective minigenome carrying the S gene from an enteric isolate. The minigenome was efficiently replicated in trans and packaged by the helper virus, leading to the formation of true recombinant and pseudorecombinant viruses containing the S proteins of both enteric and respiratory TGEV strains in their envelopes. The recombinants acquired an enteric tropism, and their analysis showed that they were generated by homologous recombination that implied a double crossover in the S gene resulting in replacement of most of the respiratory, attenuated strain S gene (nucleotides 96 to 3700) by the S gene of the enteric, virulent isolate. The recombinant virus was virulent and rapidly evolved in swine testis cells by the introduction of point mutations and in-phase codon deletions in a domain of the S gene (nucleotides 217 to 665) previously implicated in the tropism of TGEV. The helper virus, with an original respiratory tropism, was also found in the enteric tract, probably because pseudorecombinant viruses carrying the spike proteins from the respiratory strain and the enteric virus in their envelopes were formed. These results demonstrated that a change in the tropism and virulence of TGEV can be engineered by sequence changes in the S gene.
Epithelial hair follicle stem cells (eHFSCs) are required to generate, maintain and renew the continuously cycling hair follicle (HF), supply cells that produce the keratinized hair shaft and aid in the reepithelialization of injured skin. Therefore, their study is biologically and clinically important, from alopecia to carcinogenesis and regenerative medicine. However, human eHFSCs remain ill defined compared to their murine counterparts, and it is unclear which murine eHFSC markers really apply to the human HF. We address this by reviewing current concepts on human eHFSC biology, their immediate progeny and their molecular markers, focusing on Keratin 15 and 19, CD200, CD34, PHLDA1, and EpCAM/Ber-EP4. After delineating how human eHFSCs may be selectively targeted experimentally, we close by defining as yet unmet key challenges in human eHFSC research. The ultimate goal is to transfer emerging concepts from murine epithelial stem cell biology to human HF physiology and pathology.
Inflammation-associated, irreversible damage to epithelial stem cells (eSCs) of the hair follicle in their immunologically privileged niche lies at the heart of scarring alopecia, which causes permanent difficult-to-treat hair loss. We propose that the two most common and closely related forms, lichen planopilaris (LPP) and frontal fibrosing alopecia (FFA), provide excellent model diseases for studying the biology and pathology of adult human eSCs in an easily accessible human mini-organ. Emphasising the critical roles for interferon (IFN)-γ and peroxisome proliferator-activated receptor (PPAR)-γ-mediated signalling in immune privilege (IP) collapse and epithelial-mesenchymal transition (EMT) of these eSCs respectively, we argue that these pathways deserve therapeutic targeting in the future management of LPP/FFA and other eSC diseases associated with IP collapse and EMT.
The transcription regulatory sequences (TRSs) of the coronavirus transmissible gastroenteritis virus (TGEV) have been characterized by using a helper virus-dependent expression system based on coronavirusderived minigenomes to study the synthesis of subgenomic mRNAs. The TRSs are located at the 5 end of TGEV genes and include a highly conserved core sequence (CS), 5-CUAAAC-3, that is essential for mediating a 100-to 1,000-fold increase in mRNA synthesis when it is located in the appropriate context. The relevant sequences contributing to TRS activity have been studied by extending the CS 5 upstream and 3 downstream. Sequences from virus genes flanking the CS influenced transcription levels from moderate (10-to 20-fold variation) to complete mRNA synthesis silencing, as shown for a canonical CS at nucleotide (nt) 120 from the initiation codon of the S gene that did not lead to the production of the corresponding mRNA. An optimized TRS has been designed comprising 88 nt from the N gene TRS, the CS, and 3 nt 3 to the M gene CS. Further extension of the 5-flanking nucleotides (i.e., by 176 nt) decreased subgenomic RNA levels. The expression of a reporter gene (-glucuronidase) by using the selected TRS led to the production of 2 to 8 g of protein per 10 6 cells. The presence of an appropriate Kozak context led to a higher level of protein expression. Virus protein levels were shown to be dependent on transcription and translation regulation.Transmissible gastroenteritis virus (TGEV) is a member of the family Coronaviridae, which, together with the family Arteriviridae, forms the order Nidovirales (6). The coronavirus RNA genome has a length ranging from 27.6 to 31.5 kb. About two-thirds of the entire RNA comprises open reading frames (ORFs) 1a and 1ab, encoding the replicase gene. The 3Ј onethird of the genome comprises the genes encoding the structural and nonstructural proteins. The organization of the non-ORF 1 nonstructural protein genes, which are interspersed between the known structural protein genes, varies significantly among different coronavirus strains (5).Coronavirus transcription is based on RNA-dependent RNA synthesis. Coronavirus mRNAs consist of six to eight types of various sizes, depending on the coronavirus strain. The largest mRNA is the genomic RNA, which also serves as the mRNA for ORFs 1a and 1ab; the others are subgenomic mRNAs (sgmRNAs), composed of noncontiguous sequences of the parental genome. The mRNAs and the genomic RNA form a nested set of RNAs of different lengths with common 5Ј and 3Ј ends. Except for the smallest mRNA, all of the mRNAs are structurally polycistronic; in general, however, only the 5Ј-most ORF (not present in the next smallest mRNA) is translated. However, there are exceptions: some mRNAs, e.g., mRNA 5 of mouse hepatitis virus (MHV), mRNA 3 of infectious bronchitis virus, and the bovine coronavirus (BCoV) nucleocapsid mRNA, are translated by internal initiation into two or three proteins (21,25). All the mRNAs possess a 5Ј sequence of 70 to 90 nucleotides (nt) that ...
The sequences involved in the replication and packaging of transmissible gastroenteritis virus (TGEV) RNA have been studied. The structure of a TGEV defective interfering RNA of 9.7 kb (DI-C) was described previously (A. Mendez, C. Smerdou, A. Izeta, F. Gebauer, and L. Enjuanes, Virology 217: 495–507, 1996), and a cDNA with the information to encode DI-C RNA was cloned under the control of the T7 promoter. The molecularly cloned DI-C RNA was replicated intrans upon transfection of helper virus-infected cells and inhibited 20-fold the replication of the parental genome. A collection of 14 DI-C RNA deletion mutants (TGEV minigenomes) was synthetically generated and tested for their ability to be replicated and packaged. The smallest minigenome (M33) that was replicated by the helper virus and efficiently packaged was 3.3 kb. A minigenome of 2.1 kb (M21) was also replicated, but it was packaged with much lower efficiency than the M33 minigenome, suggesting that it had lost either the sequences containing the main packaging signal or the required secondary structure in the packaging signal due to alteration of the flanking sequences. The low packaging efficiency of the M21 minigenome was not due to minimum size restrictions. The sequences essential for minigenome replication by the helper virus were reduced to 1,348 nt and 492 nt at the 5′ and 3′ ends, respectively. The TGEV-derived RNA minigenomes were successfully expressed following a two-step amplification system that couples pol II-driven transcription in the nucleus to replication supported by helper virus in the cytoplasm, without any obvious splicing. This system and the use of the reporter gene β-glucuronidase (GUS) allowed minigenome detection at passage zero, making it possible to distinguish replication efficiency from packaging capability. The synthetic minigenomes have been used to design a helper-dependent expression system that produces around 1.0 μg/106 cells of GUS.
Only anagen human hair follicles show CD34 immunoreactivity. CD34 and CK15 recognize different types of cells or cells at different stages of differentiation.
Epigenetic clocks for mice were generated based on deep-sequencing analysis of the methylome. Here, we demonstrate that site-specific analysis of DNA methylation levels by pyrosequencing at only three CG dinucleotides (CpGs) in the genes Prima1, Hsf4, and Kcns1 facilitates precise estimation of chronological age in murine blood samples, too. DBA/2 mice revealed accelerated epigenetic aging as compared to C57BL6 mice, which is in line with their shorter life-expectancy. The three-CpG-predictor provides a simple and cost-effective biomarker to determine biological age in large intervention studies with mice.
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