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Reverse genetics and cystic fibrosis.

The protein responsible for cystic fibrosis has been identified using an approach called "reverse" genetics. This approach relies on the chromosomal map position to direct the search for a disease gene, several novel cloning strategies to isolate the gene, and the gene's sequence to define the abnormal protein. Reverse genetics, because it does not require prior knowledge of the protein's biochemical function, has wide utility and is being used to define the defects in many single-gene disorders. This update presents the reverse genetics approach and uses cystic fibrosis to illustrate the principles involved.

Chromosome Mapping↗

Construction of Reverse Genetics System for Feline Calicivirus FCV-BJ616 and Proteomic Analysis.

Feline calicivirus (FCV) is a primary cause of upper respiratory tract infections and oral ulcerative disease in cats and exhibits substantial genetic diversity that complicates prevention and control. In this study, we isolated the FCV-BJ616 strain, established a reverse-genetics system, and investigated its pathogenic mechanisms, thereby providing a foundation for antibody-based therapies and broad-spectrum vaccine development. The virus was purified by three rounds of plaque cloning, and its morphology was examined by electron microscopy. VP1 expression was confirmed by immunofluorescence and Western blotting. Using integrated systems-biology and reverse-genetics approaches, an infectious clone of rFCV-BJ616 was successfully assembled and rescued, exhibiting genetic stability comparable to that of the parental strain. In vivo infection experiments showed that rFCV-BJ616 retained wild-type virulence, causing persistent high fever, weight loss, and multiorgan pathology in infected cats. Proteomic analysis indicated that infection with FCV-BJ616 or rFCV-BJ616 markedly activated cytokine-mediated inflammatory signaling pathways. Both FCV-BJ616 and rFCV-BJ616 significantly upregulated the expression of IL-8, S100A8/A9, and TLR3, which are associated with acute inflammation and tissue damage. Furthermore, elevated IFN-β levels concomitant with STAT1 downregulation suggested a transient attenuation of antiviral signaling during early immune activation. These findings were corroborated by ELISA-based validation of serum cytokine profiles. Collectively, this study provides new insights into the molecular pathogenesis and evolution of FCV-BJ616 and establishes a robust reverse-genetics platform for precise genome manipulation and future vaccine development.

Animals↗

Reverse genetic studies of the DNA damage response in the chicken B lymphocyte line DT40.

In the 'post-genome' era, reverse genetics is one of the most informative and powerful means to investigate protein function. The chicken B lymphocyte line DT40 is widely used for reverse genetics because the cells have a number of advantages, including efficient gene targeting as well as a remarkably stable phenotype. Furthermore, the absence of functional p53 in DT40 cells enables identification of DNA damage using chromosome analysis by suppressing damage-induced apoptosis during interphase. This review summarizes the contribution of DT40 cells to reverse genetic studies of DNA damage response pathways in higher eukaryotic cells.

Animals↗

TILLING without a plough: a new method with applications for reverse genetics.

TILLING (Targeting Induced Local Lesions IN Genomes) is a powerful reverse genetic technique that employs a mismatch-specific endonuclease to detect induced or natural DNA polymorphisms. Its advantages over other reverse genetic techniques include its applicability to virtually any organism, its facility for high-throughput and its independence of genome size, reproductive system or generation time. TILLING is currently being used for the detection of both induced and natural variation in several plant species.

Agriculture↗

Forward and reverse genetic approaches to the analysis of eye development in zebrafish.

The zebrafish has been established as a mainstream research system, largely due to the immense success of genetic screens. Over 2000 mutant alleles affecting zebrafish's early development have been isolated in two large-scale morphological screens and several smaller efforts. So far, over 50 mutant strains display retinal defects and many more have been shown to affect the retinotectal projection. More recently, mutant isolation and characterization have been successfully followed by candidate and positional cloning of underlying genes. To supplement forward genetic mutational analysis, several reverse genetic techniques have also been developed. These recent advances, combined with the genome project, have established the zebrafish as one of the leading models for studies of visual system development.

Animals↗

A reverse genetics approach for recovery of recombinant influenza B viruses entirely from cDNA.

The recovery of recombinant influenza A virus entirely from cDNA was recently described (9, 19). We adapted the technique for engineering influenza B virus and generated a mutant bearing an amino acid change E116G in the viral neuraminidase which was resistant in vitro to the neuraminidase inhibitor zanamivir. The method also facilitates rapid isolation of single-gene reassortants suitable as vaccine seeds and will aid further investigations of unique features of influenza B virus.

Cloning, Molecular↗

Rapid Generation of Reverse Genetics Systems for Coronavirus Research and High-Throughput Antiviral Screening Using Gibson DNA Assembly.

Coronaviruses (CoVs) pose a significant threat to human health, as demonstrated by the COVID-19 pandemic. The large size of the CoV genome (around 30 kb) represents a major obstacle to the development of reverse genetics systems, which are invaluable for basic research and antiviral drug screening. In this study, we established a rapid and convenient method for generating reverse genetic systems for various CoVs using a bacterial artificial chromosome (BAC) vector and Gibson DNA assembly. Using this system, we constructed infectious cDNA clones of coronaviruses from three genera: human coronavirus 229E (HCoV-229E) of the genus Alphacoronavirus, mouse hepatitis virus A59 (MHV-59) of Betacoronavirus, and porcine deltacoronavirus (PDCoV-Haiti) of Deltacoronavirus. Since beta coronaviruses including severe acute respiratory syndrome coronavirus (SARS-CoV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and Middle East respiratory syndrome coronavirus (MERS-CoV) represent major human pathogens, we modified the infectious clone of the beta coronavirus MHV-A59 by replacing its NS5a gene with a fluorescent reporter gene to create a system suitable for high-throughput drug screening. Thus, this study provides a practical and cost-effective approach to developing reverse genetics platforms for CoV research and antiviral drug screening.

Reverse Genetics↗

Combinatorial genome engineering of pseudorabies virus Bartha by developing a reverse genetic system based on three overlapping genomic segments.

INTRODUCTION: The 138-kilobase genome of pseudorabies virus vaccine strain Bartha K61 harbors many nonessential genes for replication and exhibits remarkable capacity for incorporating foreign genes for therapeutic applications. However, the large size of the Bartha genome complicates its efficient engineering. OBJECTIVES: Development of a reverse genetic system for pseudorabies virus Bartha based on three overlapping genomic segments to facilitate multiplex genome engineering. METHODS: The 138-kb genome of Bartha was split into three overlapping segments (42 kb, 43 kb, and 53 kb), each cloned in a bacterial artificial chromosome (BAC) to facilitate genome engineering. The infectious virus was reconstituted by transfecting the 3 genomic fragments released from the BACs into Vero cells in which a complete virus genome was assembled using 2-kb overlaps between adjacent pieces. RESULTS: Employing the reverse genetic system, we individually deleted 15 candidate nonessential genes and confirmed that 10 were dispensable for viral growth in cell culture. Deletion of 7 nonessential genes had no impact on viral growth, whereas UL47 deletion reduced viral growth rate and deletions of UL44, UL47, or US3 resulted in smaller viral plaques. A total of 45 viral genomes with double deletions of nonessential genes were constructed, among which 22 were successfully rescued into infectious virions. Fifteen double-deletion mutant viruses had a viral titer comparable with the wild-type Bartha, while the remaining 7 showed a lower titer. Additionally, expressions of the mNeonGreen reporter gene at nonessential gene loci were evaluated. Cells infected with recombinant viruses carrying mNeonGreen at 8 loci showed strong green fluorescence, whereas those with mNeonGreen at 2 loci exhibited very weak fluorescence. CONCLUSION: The reverse genetic system developed in this study enables rapid and combinatorial engineering of viruses with the large DNA genome, and will accelerate development of large DNA virus-based therapeutics including live-attenuated vaccines, vector vaccines, and oncolytic herpesviruses.

Herpesvirus 1, Suid↗

Chemical- and irradiation-induced mutants of indica rice IR64 for forward and reverse genetics.

IR64, the most widely grown indica rice in South and Southeast Asia, possesses many positive agronomic characteristics (e.g., wide adaptability, high yield potential, tolerance to multiple diseases and pests, and good eating quality,) that make it an ideal genotype for identifying mutational changes in traits of agronomic importance. We have produced a large collection of chemical and irradiation-induced IR64 mutants with different genetic lesions that are amenable to both forward and reverse genetics. About 60,000 IR64 mutants have been generated by mutagenesis using chemicals (diepoxybutane and ethylmethanesulfonate) and irradiation (fast neutron and gamma ray). More than 38,000 independent lines have been advanced to M4 generation enabling evaluation of quantitative traits by replicated trials. Morphological variations at vegetative and reproductive stages, including plant architecture, growth habit, pigmentation and various physiological characters, are commonly observed in the four mutagenized populations. Conditional mutants such as gain or loss of resistance to blast, bacterial blight, and tungro disease have been identified at frequencies ranging from 0.01% to 0.1%. Results from pilot experiments indicate that the mutant collections are suitable for reverse genetics through PCR-detection of deletions and TILLING. Furthermore, deletions can be detected using oligomer chips suggesting a general technique to pinpoint deletions when genome-wide oligomer chips are broadly available. M4 mutant seeds are available for users for screening of altered response to multiple stresses. So far, more than 15,000 mutant lines have been distributed. To facilitate broad usage of the mutants, a mutant database has been constructed in the International Rice Information System (IRIS; http: //www.iris.irri.org) to document the phenotypes and gene function discovered by users.

DNA, Plant↗

Reverse genetics systems for the generation of segmented negative-sense RNA viruses entirely from cloned cDNA.

Reverse genetics is defined as the generation of virus entirely from cloned cDNA. For negative-sense RNA viruses, whose genomes are complementary to mRNA in their orientation, the viral RNA(s) and the viral proteins required for replication and translation must be provided to initiate the viral replication cycle. Segmented negative-sense RNA viruses were refractory to genetic manipulation until 1989. In this chapter, we review developments in the reverse genetics of segmented negative-sense RNA viruses, beginning with the in vitro reconstitution of viral polymerase complexes in the late 1980s and culminating in the generation of Bunyamwera and influenza virus entirely from plasmid DNA almost a decade later.

DNA, Complementary↗

Transgenesis and reverse genetics of mosquito innate immunity.

In recent years, mosquito molecular biology has been a scene of astounding achievements, namely the development of genetic transformation, characterization of inducible tissue-specific promoters, and acquirement of mosquito genome sequences. However, the lack of a complete genetic tool box for mosquitoes remains a serious obstacle in our ability to study essential mosquito-specific mechanisms. Unlike Drosophila, very few null mutations for mosquito genes exist. The development of reverse-genetic analyses based on RNAi and transgenic techniques will help to compensate for these deficiencies and aid in identification of critical genes in important regulatory pathways. The study of mosquito innate immunity is one example and described here. In this study, we combine mosquito transgenesis with reverse genetics. The advantage of transgenesis is the ability to establish genetically stable, dominant-negative and overexpression phenotypes. Using the blood-meal-activated vitellogenin gene (Vg) promoter, we have generated transgenic mosquitoes with blood-meal-activated, overexpressed antimicrobial peptides, Defensin A and Cecropin A. Moreover, we have recently generated a transgenic dominant-negative Relish mosquito strain, which after taking a blood meal, becomes immune-deficient to infection by Gram-negative bacteria. The latter accomplishment has opened the door to a reverse-genetic approach in mosquitoes based on transgenesis.

Animals↗

Reverse genetics by fast neutron mutagenesis in higher plants.

Fast neutron mutagenesis has long been used for forward genetic studies in plants. Only recently has it been used for reverse genetics. By combining fast neutron mutagenesis and high throughput PCR screening, a new knockout methodology, Deleteagene (Delete-a-gene), was developed in Arabidopsis and rice to obtain deletion mutants for target genes. Since fast neutron is a highly efficient mutagen that produces deletion mutations easily detectable by PCR, this method has the potential to enable reverse genetic screens in most plant species. In this review, we will discuss the use of fast neutron as a mutagen for creating deletion mutations, and strategies for successful application of Deleteagene in other plant species.

Arabidopsis↗

A plasmid-based reverse genetics system for influenza A virus.

A reverse genetics system for negative-strand RNA viruses was first successfully developed for influenza viruses. This technology involved the transfection of in vitro-reconstituted ribonucleoprotein (RNP) complexes into influenza virus-infected cells. We have now developed a method that allows intracellular reconstitution of RNP complexes from plasmid-based expression vectors. Expression of a viral RNA-like transcript is achieved from a plasmid containing a truncated human polymerase I (polI) promoter and a ribozyme sequence that generates the desired 3' end by autocatalytic cleavage. The polI-driven plasmid is cotransfected into human 293 cells with polII-responsive plasmids that express the viral PB1, PB2, PA, and NP proteins. This exclusively plasmid-driven system results in the efficient transcription and replication of the viral RNA-like reporter and allows the study of cis- and trans-acting signals involved in the transcription and replication of influenza virus RNAs. Using this system, we have also been able to rescue a synthetic neuraminidase gene into a recombinant influenza virus. This method represents a convenient alternative to the previously established RNP transfection system.

Base Sequence↗

Generation of high-yielding influenza A viruses in African green monkey kidney (Vero) cells by reverse genetics.

Influenza A viruses are the cause of annual epidemics of human disease with occasional outbreaks of pandemic proportions. The zoonotic nature of the disease and the vast viral reservoirs in the aquatic birds of the world mean that influenza will not easily be eradicated and that vaccines will continue to be needed. Recent technological advances in reverse genetics methods and limitations of the conventional production of vaccines by using eggs have led to a push to develop cell-based strategies to produce influenza vaccine. Although cell-based systems are being developed, barriers remain that need to be overcome if the potential of these systems is to be fully realized. These barriers include, but are not limited to, potentially poor reproducibility of viral rescue with reverse genetics systems and poor growth kinetics and yields. In this study we present a modified A/Puerto Rico/8/34 (PR8) influenza virus master strain that has improved viral rescue and growth properties in the African green monkey kidney cell line, Vero. The improved properties were mediated by the substitution of the PR8 NS gene for that of a Vero-adapted reassortant virus. The Vero growth kinetics of viruses with H1N1, H3N2, H6N1, and H9N2 hemagglutinin and neuraminidase combinations rescued on the new master strain were significantly enhanced in comparison to those of viruses with the same combinations rescued on the standard PR8 master strain. These improvements pave the way for the reproducible generation of high-yielding human and animal influenza vaccines by reverse genetics methods. Such a means of production has particular relevance to epidemic and pandemic use.

Animals↗

Responsiveness to a pandemic alert: use of reverse genetics for rapid development of influenza vaccines.

BACKGROUND: In response to the emergence of severe infection capable of rapid global spread, WHO will issue a pandemic alert. Such alerts are rare; however, on Feb 19, 2003, a pandemic alert was issued in response to human infections caused by an avian H5N1 influenza virus, A/Hong Kong/213/03. H5N1 had been noted once before in human beings in 1997 and killed a third (6/18) of infected people. The 2003 variant seemed to have been transmitted directly from birds to human beings and caused fatal pneumonia in one of two infected individuals. Candidate vaccines were sought, but no avirulent viruses antigenically similar to the pathogen were available, and the isolate killed embryonated chicken eggs. Since traditional strategies of vaccine production were not viable, we sought to produce a candidate reference virus using reverse genetics. METHODS: We removed the polybasic aminoacids that are associated with high virulence from the haemagglutinin cleavage site of A/Hong Kong/213/03 using influenza reverse genetics techniques. A reference vaccine virus was then produced on an A/Puerto Rico/8/34 (PR8) backbone on WHO-approved Vero cells. We assessed this reference virus for pathogenicity in in-vivo and in-vitro assays. FINDINGS: A reference vaccine virus was produced in Good Manufacturing Practice (GMP)-grade facilities in less than 4 weeks from the time of virus isolation. This virus proved to be non-pathogenic in chickens and ferrets and was shown to be stable after multiple passages in embryonated chicken eggs. INTERPRETATION: The ability to produce a candidate reference virus in such a short period of time sets a new standard for rapid response to emerging infectious disease threats and clearly shows the usefulness of reverse genetics for influenza vaccine development. The same technologies and procedures are currently being used to create reference vaccine viruses against the 2004 H5N1 viruses circulating in Asia.

Animals↗

Evaluation of a genetically modified reassortant H5N1 influenza A virus vaccine candidate generated by plasmid-based reverse genetics.

Avian influenza A H5N1 viruses similar to those that infected humans in Hong Kong in 1997 continue to circulate in waterfowl and have reemerged in poultry in the region, raising concerns that these viruses could reappear in humans. The currently licensed trivalent inactivated influenza vaccines contain hemagglutinin (HA) and neuraminidase genes from epidemic strains in a background of internal genes derived from the vaccine donor strain, A/Puerto Rico/8/34 (PR8). Such reassortant candidate vaccine viruses are currently not licensed for the prevention of human infections by H5N1 influenza viruses. A transfectant H5N1/PR8 virus was generated by plasmid-based reverse genetics. The removal of the multibasic amino acid motif in the HA gene associated with high pathogenicity in chickens, and the new genotype of the H5N1/PR8 transfectant virus, attenuated the virus for chickens and mice without altering the antigenicity of the HA. A Formalin-inactivated vaccine prepared from this virus was immunogenic and protected mice from subsequent wild-type H5N1 virus challenge. This is the first successful attempt to develop an H5N1 vaccine seed virus resembling those used in currently licensed influenza A vaccines with properties that make it a promising candidate for further evaluation in humans.

Animals↗

Reverse genetics of Caenorhabditis elegans.

It is somewhat ironic that animals that are the prime choice for detailed genetic analysis, such as the fruit fly and the nematode, have thus far been largely refractory to reverse genetic analysis. Their detailed genetic map, and small genome size have made them subjects of ambitious genome analysis projects, but there is still no strategy to introduce desired changes into their genomes by homologous recombination. Some alternative approaches have recently become available; this review describes possibilities and unsolved problems for reverse genetics in the nematode Caenorhabditis elegans. The transposon Tc1 could prove to be very useful for the isolation of knock out mutants, and possibly also for introduction of more subtle alterations.

Animals↗

Reverse genetics system for Uukuniemi virus (Bunyaviridae): RNA polymerase I-catalyzed expression of chimeric viral RNAs.

We describe here the development of a reverse genetics system for the phlebovirus Uukuniemi virus, a member of the Bunyaviridae family, by using RNA polymerase I (pol I)-mediated transcription. Complementary DNAs containing the coding sequence for either chloramphenicol acetyltransferase (CAT) or green fluorescent protein (GFP) (both in antisense orientation) were flanked by the 5'- and 3'-terminal untranslated regions of the Uukuniemi virus sense or complementary RNA derived from the medium-sized (M) RNA segment. This chimeric cDNA (pol I expression cassette) was cloned between the murine pol I promoter and terminator and the plasmid transfected into BHK-21 cells. When such cells were either superinfected with Uukuniemi virus or cotransfected with expression plasmids encoding the L (RNA polymerase), N (nucleoprotein), and NSs (nonstructural protein) viral proteins, strong CAT activity or GFP expression was observed. CAT activity was consistently stronger in cells expressing L plus N than following superinfection. No activity was seen without superinfection, nor was activity detected when either the L or N expression plasmid was omitted. Omitting NSs expression had no effect on CAT activity or GFP expression, indicating that this protein is not needed for viral RNA replication or transcription. CAT activity could be serially passaged to fresh cultures by transferring medium from CAT-expressing cells, indicating that recombinant virus containing the reporter construct had been produced. In summary, we demonstrate that the RNA pol I system, originally developed for influenza virus, which replicates in the nucleus, has strong potential for the development of an efficient reverse genetics system also for Bunyaviridae members, which replicate in the cytoplasm.

Animals↗