This work was supported by the financial support of the doctoral research initiation funds provided by the China West Normal University (No. 20E059).

1. Preparation of the template of the PRRSV gene
<ol>
	<li>Thaw the virus stock in 1 mL of RNA extraction reagent (see Table of Materials).</li>
	<li>Add 0.2 mL of chloroform and mix thoroughly. Incubate for 3 min.</li>
	<li>Centrifuge the mixture for 15 min at 12,000 &#215; g at 4 &#176;C. 
	NOTE: The mixture is divided into three phases, namely, a colorless aqueous phase, an interphase, and a red phenol-chloroform phase.</li>
	<li>Pipet the colorless aqueous phase out and transfer it to a new tube.</li>
	<li>Mix the aqueous phase thoroughly with 0.5 mL of isopropanol and incubate for 10 min at 4 &#176;C.</li>
	<li>Centrifuge for 10 min at 12,000 &#215; g at 4 &#176;C. 
	NOTE: The bottom of the tube has a white RNA precipitate.</li>
	<li>Use a micropipette to discard the supernatant of the tube.</li>
	<li>Add 1 mL of 75% ethanol to resuspend the pellet and vortex briefly.</li>
	<li>Centrifuge for 5 min at 7,500 &#215; g at 4 &#176;C. Use a micropipette to discard the supernatant of the tube.</li>
	<li>Dry the RNA for 5 min, add 20-50 &#181;L of RNase-free water to resuspend the RNA, and mix thoroughly.</li>
	<li>Proceed to perform reverse transcription; set up the reactions to perform reverse transcription as shown in Table 1. 
	NOTE: To ensure successful reverse transcription, use high-quality RNA templates.</li>
	<li>Use the resulting cDNA for PCR or store it at -20 &#176;C.</li>
</ol>
2. PCR primer design
<ol>
	<li>Designing the forward primer
	<ol>
		<li>Open the software and choose New DNA File.</li>
		<li>Paste the PRRSV gene sequence (GenBank: FJ548852.1) from NCBI into the software. Click OK to generate the sequence files.</li>
		<li>Analyze the sequence and mark the fragment junctions. Design the forward specific sequence primer of the fragments. For most cases, the preferable melting temperature (Tm) is between 55 &#176;C and 62 &#176;C, and the GC content is 40-60%.</li>
		<li>Click on Primers and choose Add Primer.</li>
		<li>Paste the specific sequence and add overlap sequences of the vector to the first 5&#39; nucleotide of the specific sequence primer. Near each junction, choose 20-40 bp to serve as the overlap region between the two adjacent fragments.</li>
		<li>Name the forward primer containing the overhangs and the specific sequence (see Supplemental Figure S1).</li>
		<li>Click on Add Primer to Template.</li>
	</ol>
	</li>
	<li>Designing the reverse primer
	<ol>
		<li>Analyze the sequence and mark the fragment junctions in the software. Design the reverse specific sequence primer of the fragments.</li>
		<li>Click on Primers and choose Add Primer.</li>
		<li>Paste the specific sequence and add the restriction site to the first 5&#39; nucleotide of the specific sequence primer. 
		NOTE: The restriction sites added must be absent from the insert fragment and the vector except for the multiple cloning sites.</li>
		<li>Add 20-40 bp overhang sequences of vectors to the first 5&#39; nucleotide of the restriction site.</li>
		<li>Name the reverse primer containing the overhangs and the specific sequence (see Supplemental Figure S1).</li>
		<li>Click on Add Primer to Template.</li>
	</ol>
	</li>
</ol>
3. PCR to amplify fragments
<ol>
	<li>Set up six individual PCR reactions (Table 2): for fragment 1, use primers P1 and P2 (see Supplemental Figure S2); for fragment 2, use primers P3 and P4 (see Supplemental Figure S2); for fragment 3, use primers P5 and P6 (see Supplemental Figure S2); for fragment 4, use primers P7 and P8 (see Supplemental Figure S2); for fragment 5, use primers P9 and P10; and use primers P11 and P12 to amplify fragment 6 (see Supplemental Figure S2). 
	NOTE: Thaw, mix, and briefly centrifuge each component before use.</li>
	<li>Run the PCR using the three-step protocol in Table 2.</li>
	<li>Add 1&#160;&#181;L of 6x DNA loading buffer to 5&#160;&#181;L of PCR product, mix, and briefly centrifuge the contents.</li>
	<li>Analyze the samples using 1% agarose gel electrophoresis.</li>
</ol>
4. Purification of the PCR fragments
NOTE: Purifying the PCR products from a gel using a gel extraction kit (see Table of Materials) is important for vector construction.
<ol>
	<li>Add 9 &#181;L of 6x DNA loading buffer to 45 &#181;L of PCR products, mix, and briefly centrifuge the contents.</li>
	<li>Perform 1% agarose gel/goldview electrophoresis to separate the DNA fragments. 
	NOTE: Do not reuse TAE running buffer as its pH value will affect DNA fragment recovery.</li>
	<li>Upon adequate separation of bands, use a sharp scalpel to carefully excise the DNA bands. 
	NOTE: Minimize the size of the gel slice by trimming off excess agarose.</li>
	<li>Weigh the gel slice in a clean 1.5 mL microcentrifuge tube, add an equal volume of binding buffer to the gel slice (e.g., 0.3 mL to a 0.3 g slice), and incubate at 60 &#176;C for 7 min.</li>
	<li>Insert a mini column in a 2 mL collection tube. Add 700 &#181;L of DNA/agarose solution from step 4.4 to the mini column.</li>
	<li>Centrifuge at 10,000 &#215; g for 1 min at room temperature. Discard the filtrate and reuse the collection tube.</li>
	<li>Add 300 &#181;L of binding buffer and centrifuge at 13,000 &#215; g for 1 min at room temperature. Discard the filtrate and reuse the collection tube.</li>
	<li>Add 700 &#181;L of wash buffer and centrifuge at 13,000 &#215; g for 1 min at room temperature. Discard the filtrate and reuse the collection tube.</li>
	<li>Spin the empty mini column for 2 min at maximum speed to dry the column matrix. Place the mini column in a clean 1.5 mL microcentrifuge tube.</li>
	<li>Add 20 &#181;L of deionized water directly to the center of the column membrane and let sit at room temperature for 2 min. Centrifuge at 13,000 &#215; g speed for 1 min.</li>
	<li>Store the DNA at -20 &#176;C.</li>
</ol>
5. Preparation of a linearized vector
NOTE: After preparing the plasmid, the selected enzymes can be used to cut it. Long digestion or dual enzyme digestion is crucial for ensuring the digestion of all DNA. This will reduce the number of false-positive clones in subsequent experiments.
<ol>
	<li>pVAX1 linearization
	<ol>
		<li>Prepare the reaction mixture at room temperature in the order indicated (Table 3). 
		NOTE: The volume of water should be added to keep the indicated total reaction volume. Here, 1 &#181;g of the plasmid was digested with enzymes in the reaction mixture. Depending on the plasmid concentration, the volume of the plasmid can be adjusted in the reaction mixture.</li>
		<li>Mix gently; then spin down. Incubate at 37 &#176;C in a heat block or water thermostat for 60 min.</li>
		<li>Perform gel purification similar to the purification of the PCR fragments in section 4 using the gel extraction kit (see Table of Materials).</li>
	</ol>
	</li>
	<li>pVAX1-F1 linearization 
	NOTE: pVAX1-F1 is the vector obtained from the first round of pVAX1 (NdeI and HindIII) and fragment 1 recombination. pVAX1-F2 is the vector obtained from the round of pVAX1-F1 (NdeI) and fragment 2 recombination. pVAX1-F3 is the vector obtained from the round of pVAX1-F2 (NdeI) and fragment 3 recombination. pVAX1-F4 is the vector obtained from the round of pVAX1-F3 (NdeI) and fragment 4 recombination. pVAX1-F5 is the vector obtained from the round of pVAX1-F4 (EcoRV and NtoI) and fragment 5 recombination.
	<ol>
		<li>For each vector, prepare the reaction mixture separately at room temperature in the order indicated (Table 3).</li>
		<li>Mix gently; then spin down. Incubate at 37 &#176;C in a heat block or water thermostat for 60 min.</li>
		<li>Perform gel purification similar to the purification of the PCR fragments in section 4 using the gel extraction kit (see Table of Materials).</li>
	</ol>
	</li>
</ol>
6. Subcloning to a new vector
NOTE: Good cloning efficiency can be achieved when using 50-200 ng of vector and inserts.
<ol>
	<li>Set up the ExonArt seamless cloning and assembly reaction (Table 4). Adjust the total reaction volume to 10 &#181;L using sterilized deionized H2O and mix.</li>
	<li>Incubate the reaction in a thermocycler for 15-60 min at 50 &#176;C. Store the samples on ice. 
	NOTE: Extending the incubation up to 60 min may enhance assembly efficiency.</li>
	<li>Thaw DH5&#945; chemically competent cells on ice.</li>
	<li>Add 10 &#181;L of the assembly product to the competent cells; then, mix gently by pipetting up and down.</li>
	<li>Place the mixture on ice for 30 min.</li>
	<li>Heat shock at 45 &#176;C for 45 s and transfer the tubes onto ice for 3 min.</li>
	<li>Add 900 &#181;L of SOC medium to the tube.</li>
	<li>Shake the tube at 225 rpm for 1 h in a 37 &#176;C shaking incubator.</li>
	<li>Centrifuge the transformation reaction at 6,000 &#215; g for 2 min. Discard the supernatant and resuspend the cells in 100 &#181;L of fresh SOC medium.</li>
	<li>Spread the transformed cell suspension on a separate LB plate with 50 &#181;g/mL kanamycin.</li>
	<li>Incubate all plates overnight at 37 &#176;C. Pick individual isolated colonies from each experimental plate.</li>
</ol>
7. Analyzing the transformants
<ol>
	<li>Pick eight colonies into 20 &#181;L of LB medium containing 50 &#181;g/mL kanamycin.</li>
	<li>Set up the colony PCR reaction and run the PCR (Table 5). 
	NOTE: For the colony PCR reaction for fragment 1, use primers P1 and P2 (see Supplemental File 1); for fragment 2, use primers P3 and P4 (see Supplemental File 1); for fragment 3, use primers P5 and P6 (see Supplemental File 1); for fragment 4, use primers P7 and P8 (see Supplemental Figure S2); for fragment 5, use primers P9 and P10; and use primers P11 and P12 to detect fragment 6 (see Supplemental Figure S2).</li>
	<li>Add 1 &#181;L of 6x DNA loading buffer to 5 &#181;L of the PCR product, mix, and briefly centrifuge the contents.</li>
	<li>Analyze the results using 1% agarose gel electrophoresis.</li>
	<li>Select one positive colony into 5 mL of LB medium containing 50 &#181;g/mL kanamycin and grow overnight at 37 &#176;C.</li>
	<li>Obtain the plasmid using a plasmid DNA mini kit (see Table of Materials) according to the manufacturer&#39;s instructions.</li>
	<li>Visualize the results using 1% agarose gel electrophoresis.</li>
</ol>

Cloning the Full-Length PRRSV Gene in the pVAX1 Expression Vector

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The authors declare no conflicts of interest.

The Gibson assembly technique is an in vitro recombination-based molecular cloning method for the assembly of DNA fragments8. This method enables the assembly of multiple DNA fragments into a circular plasmid in a single-tube isothermal reaction. However, one of the obstacles to the Gibson Assembly technique is the acquisition of long fragments from cDNA. The long fragments are difficult to accurately amplify for many reasons. For example, primers are easier to mismatch during long extend...

As an essential technique to construct DNA-based experimental tools for expression in prokaryotic and eukaryotic cells, molecular cloning is a very important component of experimental biology. Molecular cloning involves four processes: the acquisition of insert DNA, ligation of the insert into the appropriate vector, transformation of the recombinant vector into Escherichia coli (E. coli), and identification of the positive clones1. So far, multiple methods have been adopted for joining DNA molecules by using restriction enzymes2,3 and PCR-mediated recombination4,5,6. Homologous recombination, known as seamless cloning technology, is the group of cloning methods, which allows sequence-independent and scarless insertion of one or more fragments of DNA into a vector. This technology includes sequence- and ligation-independent cloning (SLIC), Seamless Ligation Cloning Extract (SLiCE), In-Fusion, and Gibson Assembly. It employs an exonuclease to degrade one strand of the insert and a vector to generate cohesive ends, and either in vivo repair or in vitro recombination to covalently join the insert to the vector by forming phosphodiester bonds. The ability to join a single insert to a vector at any sequence without any scars is very appealing. Furthermore, the technology has the ability to join 5-10 fragments in a predetermined order without sequence restrictions.
As one of many recombinant DNA techniques, the Gibson Assembly technique, currently the most effective cloning method7,8, is a robust and elegant exonuclease-based method to assemble one or multiple linearized DNA fragments seamlessly. The Gibson Assembly reaction is performed under isothermal conditions using a mixture of three enzymes,namely, 5&#39; exonuclease, high-fidelity polymerase, and a thermostable DNA ligase. Single-strand 3&#180; overhangs created by the 5&#39;-3&#39; exonuclease contribute to the annealing of fragments that share complementarity at one end. The high-fidelity polymerase effectively fills the gaps in the annealed single-strand regions by adding dNTPs, and the thermostable DNA ligase seals the nicks to form joint DNA molecules8. Hence, this technical method has been widely used for the construction of gene expression vectors.
Porcine reproductive and respiratory syndrome (PRRS) is a viral disease that leads to reproductive impairment and respiratory failure in pigs caused by PRRSV at any age9. The syndrome is manifested as fever, anorexia, pneumonia, lethargy, depression, and respiratory distress. Moreover, clinical signs, including red/blue discoloration of the ears, have been observed in some epidemics. As a member of the family arterivirus, PRRSV is widely transmitted to pork-producing countries by direct contact and exchange of fluids, including urine, colostrum, and saliva. Due to the spread of PRRSV in the United States, the total economic losses of the pork industry have been estimated to be approximately $664 million per year, based on the breeding scale of 5.8 million sows and 110 million pigs10,11. The Animal and Plant Health Inspection Service report shows that 49.8% of unvaccinated pigs show the presence of PRRSV in serum12 and low levels of PRRSV in infected pigs are excreted through saliva, nasal secretions, urine, and feces13. Multiple strategies have been implemented to control PRRSV propagation14,15,16. In addition to elimination procedures to create completely virus-negative populations or improving biosafety and management, administering vaccines is an effective means of controlling PRRS.
PRRSV is an enveloped, single-stranded, positive-sense RNA virus with a length of approximately 15 kilobases (kb). The PRRSV genome consists of at least 10 open reading frames (ORFs), a short 5&#39; untranslated region (5&#39; UTR), and a poly(A) tail at the 3&#39; terminus (Figure 1A)17. The genome of a negative-stranded RNA virus is non-infectious whereas the genome from positive-stranded RNA viruses is infectious. There are two main strategies for RNA and DNA transfection for generating virus progeny18. However, cloning the full-length fragment corresponding to the RNA genome is crucial for the construction of infectious clones. Due to the long and complex nature of the PRRSV genome, the full-length genome cannot be easily obtained through PCR at once. Additionally, although the artificial synthesis of PRRSV genes is an effective solution, the synthesis of long fragments is often expensive. Hence, to obtain the PRRSV full-length expression vector, we attempted to create it by the multiple inserts homologous recombination method19,20. Unfortunately, we were not able to obtain the full-length gene expression vector. Therefore, in this study, we added appropriate restriction sites to the reverse primer and successfully obtained the pVAX1-PRRSV expression vector by several rounds of homologous recombination reactions. Furthermore, this method can also achieve deletion or mutation of target genes and efficiently join a large number of DNA fragments to the expression vector.

<table><tbody><tr><td>1 kb plus DNA Ladder</td><td>Tiangen Biochemical Technology (Beijing) Co., Ltd</td><td>MD113-02</td><td></td></tr><tr><td>2x Universal Green PCR Master Mix</td><td>Rong Wei Gene Biotechnology Co., Ltd</td><td>A303-1</td><td></td></tr><tr><td>Agarose</td><td>Sangon Biotech (Shanghai) Co., Ltd.</td><td>9012-36-6</td><td></td></tr><tr><td>Benchtop Microcentrifuge</td><td>Thermo Fisher Scientific&amp;nbsp; Co., Ltd</td><td>FRESCO17</td><td></td></tr><tr><td>Clean Bench</td><td>Sujing Antai Air Technology&amp;nbsp; Co., Ltd</td><td>VD-650-U</td><td></td></tr><tr><td>DNA Electrophoresis Equipment</td><td>Cleaver Scientific&amp;nbsp; Co., Ltd</td><td>170905117</td><td></td></tr><tr><td>DNA Loading Buffer (6x)</td><td>Biosharp Biotechnology Co., Ltd</td><td>BL532A</td><td></td></tr><tr><td>E. Z. N. A. Gel Extraction kit</td><td>Omega Bio-Tek Co., Ltd</td><td>D2500-01</td><td></td></tr><tr><td>E.Z.N.A. Plasmid DNA Mini Kit I</td><td>Omega Bio-Tek Co., Ltd</td><td>D6943-01</td><td></td></tr><tr><td>Electro-heating Standing-temperature Cultivator</td><td>Shanghai Hengyi Scientific Instrument Co., Ltd</td><td>DHP-9082</td><td></td></tr><tr><td>ExonArt Seamless Cloning and Assembly kit</td><td>Rong Wei Gene Biotechnology Co., Ltd</td><td>A101-02</td><td></td></tr><tr><td>ExonScript RT SuperMix with dsDNase</td><td>Rong Wei Gene Biotechnology Co., Ltd</td><td>A502-1</td><td></td></tr><tr><td>FastDigest Eco321 (EcoRV)</td><td>Thermo Fisher Scientific&amp;nbsp; Co., Ltd</td><td>FD0303</td><td></td></tr><tr><td>FastDigest HindIII</td><td>Thermo Fisher Scientific&amp;nbsp; Co., Ltd</td><td>FD0504</td><td></td></tr><tr><td>FastDigest NheI</td><td>Thermo Fisher Scientific&amp;nbsp; Co., Ltd</td><td>FD0974</td><td></td></tr><tr><td>FastDigest NotI</td><td>Thermo Fisher Scientific&amp;nbsp; Co., Ltd</td><td>FD0596</td><td></td></tr><tr><td>Gel Doc XR</td><td>Bio-Rad Laboratories Co., Ltd</td><td>721BR07925</td><td></td></tr><tr><td>Goldview Nucleic Acid Gel Stain</td><td>Shanghai Yubo Biotechnology Co., Ltd</td><td>YB10201ES03</td><td></td></tr><tr><td>Ice Maker Machine</td><td>Shanghai Bilang Instrument Manufacturing Co., Ltd</td><td>FMB100</td><td></td></tr><tr><td>Invitrogen Platinum SuperFi II DNA Polymerase</td><td>Thermo Fisher Scientific&amp;nbsp; Co., Ltd</td><td>12361010</td><td></td></tr><tr><td>LB Agar Plate (Kanamycin)</td><td>Sangon Biotech (Shanghai) Co., Ltd.</td><td>B530113-0010</td><td></td></tr><tr><td>LB sterile liquid medium (Kanamycin)</td><td>Sangon Biotech (Shanghai) Co., Ltd.</td><td>B540113-0001</td><td></td></tr><tr><td>Micropipettors</td><td>Thermo Fisher Scientific&amp;nbsp; Co., Ltd</td><td>&amp;mdash;</td><td></td></tr><tr><td>Microwave Oven</td><td>Panasonic Electric (China) Co., Ltd</td><td>NN-GM333W</td><td></td></tr><tr><td>Orbital Shakers</td><td>Shanghai Zhicheng Analytical Instrument Manufacturing Co., Ltd</td><td>ZHWY-2102C</td><td></td></tr><tr><td>PRRSV virus</td><td>Sichuan Agricultural University</td><td>&amp;mdash;</td><td></td></tr><tr><td>SnapGene</td><td>GSL Biotech, LLC</td><td>v5.1</td><td>To design primers</td></tr><tr><td>T100 PCR Gradient Thermal Cycler</td><td>Bio-Rad Laboratories&amp;nbsp; Co., Ltd</td><td>T100 Thermal Cycler</td><td></td></tr><tr><td>TAE buffer</td><td>Sangon Biotech (Shanghai) Co., Ltd.</td><td>B040123-0010</td><td></td></tr><tr><td>TRIzol Reagent</td><td>Thermo Fisher Scientific&amp;nbsp; Co., Ltd</td><td>15596026</td><td>RNA extraction reagent</td></tr></tbody></table>

a seamless cloning approach for porcine reproductive and respiratory syndrome virus expression vector construction

The construction of gene expression vectors is an important component of laboratory work in experimental biology. With technical advancements like Gibson Assembly, vector construction becomes relatively simple and efficient. However, when the full-length genome of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) cannot be easily amplified by a single polymerase chain reaction (PCR) from cDNA, or it is difficult to acquire a full-length gene expression vector by homologous recombination of multiple inserts in vitro, the current Gibson Assembly technique fails to achieve this goal.
Consequently, we aimed to divide the PRRSV genome into several fragments and introduce appropriate restriction sites into the reverse primer for obtaining PCR-amplified fragments. After joining the previous DNA fragment into the vector by homologous recombination technology, the new vector acquired the restriction enzyme cleavage site. Thus, we can linearize the vector by using the newly added enzyme cleavage site and introduce the next DNA fragment downstream of the upstream DNA fragment.
The introduced restriction enzyme cleavage site at the 3&#39; end of the upstream DNA fragment will be eliminated, and a new cleavage site will be introduced into the 3&#39; end of the downstream DNA fragment. In this way, we can join DNA fragments to the vector one by one. This method is applicable to successfully construct the PRRSV expression vector and is an effective method for assembling a large number of fragments into the expression vector.

In this paper, we present an in vitro recombination system to assemble and repair overlapping DNA molecules using the reverse primer via continually introduced restriction sites (Figure 1B). This system is a simple and efficient procedure comprising the preparation of the linear vector and the insert fragments containing overhangs introduced by PCR with primers having appropriate 5&#39; extension sequences and restriction sites; an in vitro single isothermal reaction and th...

Author Spotlight: Exploring Cloning Techniques for Full-Length DNA Fragments

Here, we present a protocol to obtain the pVAX1-PRRSV expression vector by introducing suitable restriction sites at the 3' end of the inserts. We can linearize the vector and join DNA fragments to the vector one by one through homologous recombination technology.

A Seamless Cloning Approach for Porcine Reproductive and Respiratory Syndrome Virus Expression Vector Construction

The construction of gene expression vectors is an important component of laboratory work in experimental biology. With technical advancements like Gibson ...

a-seamless-cloning-approach-for-porcine-reproductive-respiratory

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

493 Views.  China West Normal University. Here, we present a protocol to obtain the pVAX1-PRRSV expression vector by introducing suitable restriction sites at the 3' end of the inserts. We can linearize the vector and join DNA fragments to the vector one by one through homologous recombination technology. 

Video: Author Spotlight: Exploring Cloning Techniques for Full-Length DNA Fragments

Using Reverse Genetics to Manipulate the NSs Gene of the Rift Valley Fever Virus MP-12 Strain to Improve Vaccine Safety and Efficacy

Rift Valley fever virus (RVFV), which causes hemorrhagic fever, neurological disorders or blindness in humans, and a high rate abortion and fetal malformation in ruminants1, has been classified as a HHS/USDA overlap select agent and a risk group 3 pathogen. It belongs to the genus Phlebovirus in the family Bunyaviridae and is one of the most virulent members of this family. Several reverse genetics systems for the RVFV MP-12 vaccine strain2,3 as well as wild-type RVFV strains 4-6, including ZH548 and ZH501, have been developed since 2006. The MP-12 strain (which is a risk group 2 pathogen and a non-select agent) is highly attenuated by several mutations in its M- and L-segments, but still carries virulent S-segment RNA3, which encodes a functional virulence factor, NSs. The rMP12-C13type (C13type) carrying 69% in-frame deletion of NSs ORF lacks all the known NSs functions, while it replicates as efficient as does MP-12 in VeroE6 cells lacking type-I IFN. NSs induces a shut-off of host transcription including interferon (IFN)-beta mRNA7,8 and promotes degradation of double-stranded RNA-dependent protein kinase (PKR) at the post-translational level.9,10 IFN-beta is transcriptionally upregulated by interferon regulatory factor 3 (IRF-3), NF-kB and activator protein-1 (AP-1), and the binding of IFN-beta to IFN-alpha/beta receptor (IFNAR) stimulates the transcription of IFN-alpha genes or other interferon stimulated genes (ISGs)11, which induces host antiviral activities, whereas host transcription suppression including IFN-beta gene by NSs prevents the gene upregulations of those ISGs in response to viral replication although IRF-3, NF-kB and activator protein-1 (AP-1) can be activated by RVFV7. . Thus, NSs is an excellent target to further attenuate MP-12, and to enhance host innate immune responses by abolishing the IFN-beta suppression function. Here, we describe a protocol for generating a recombinant MP-12 encoding mutated NSs, and provide an example of a screening method to identify NSs mutants lacking the function to suppress IFN-beta mRNA synthesis. In addition to its essential role in innate immunity, type-I IFN is important for the maturation of dendritic cells and the induction of an adaptive immune response12-14. Thus, NSs mutants inducing type-I IFN are further attenuated, but at the same time are more efficient at stimulating host immune responses than wild-type MP-12, which makes them ideal candidates for vaccination approaches.

The reverse genetics system for the Rift Valley fever virus MP-12 vaccine strain is a useful tool for creating additional MP-12 mutants with increased attenuation and immunogenicity. We describe the protocol to generate and characterize NSs mutant strains.

Rift Valley fever virus (RVFV), which causes hemorrhagic fever, neurological disorders or blindness in humans, and a high rate abortion and fetal ...

Immunology and Infection

CRISPR Guide RNA Cloning for Mammalian Systems

The outlined protocol describes streamlined methods for the efficient and cost-effective generation of Cas9-associated guide RNAs. Two alternative strategies for guide RNA (gRNA) cloning are outlined based on the usage of the Type IIS restriction enzyme BsmBI in combination with a set of compatible vectors. Outside of the access to Sanger sequencing services to validate the generated vectors, no special equipment or reagents are required aside from those that are standard to modern molecular biology laboratories. The outlined method is primarily intended for cloning one single gRNA or one paired gRNA-expressing vector at a time. This procedure does not scale well for the generation of libraries containing thousands of gRNAs. For those purposes, alternative sources of oligonucleotide synthesis such as oligo-chip synthesis are recommended. Finally, while this protocol focuses on a set of mammalian vectors, the general strategy is plastic and is applicable to any organism if the appropriate gRNA vector is available.

Here, a simple, efficient, and cost-effective method of sgRNA cloning is outlined.

The outlined protocol describes streamlined methods for the efficient and cost-effective generation of Cas9-associated guide RNAs. Two alternative ...

Genetics

Generation and Assembly of Virus-Specific Nucleocapsids of the Respiratory Syncytial Virus

The use of an authentic RNA template is critical to advance the fundamental knowledge of viral RNA synthesis that can guide both mechanistic discovery and assay development in virology. The RNA template of nonsegmented negative-sense (NNS) RNA viruses, such as the respiratory syncytial virus (RSV), is not an RNA molecule alone but rather a nucleoprotein (N) encapsidated ribonucleoprotein complex. Despite the importance of the authentic RNA template, the generation and assembly of such a ribonucleoprotein complex remain sophisticated and require in-depth elucidation. The main challenge is that the overexpressed RSV N binds non-specifically to cellular RNAs to form random nucleocapsid-like particles (NCLPs). Here, we established a protocol to obtain RNA-free N (N0) first by co-expressing N with a chaperone phosphoprotein (P), then assembling N0 with RNA oligos with the RSV-specific RNA sequence to obtain virus-specific nucleocapsids (NCs). This protocol shows how to overcome the difficulty in the preparation of this traditionally challenging viral ribonucleoprotein complex.

For in-depth mechanistic analysis of the respiratory syncytial virus (RSV) RNA synthesis, we report a protocol of utilizing the chaperone phosphoprotein (P) for coexpression of the RNA-free nucleoprotein (N0) for subsequent in vitro assembly of the virus-specific nucleocapsids (NCs).

The use of an authentic RNA template is critical to advance the fundamental knowledge of viral RNA synthesis that can guide both mechanistic discovery and ...

Biochemistry

Bacterial Artificial Chromosomes: A Functional Genomics Tool for the Study of Positive-strand RNA Viruses

Reverse genetics, an approach to rescue infectious virus entirely from a cloned cDNA, has revolutionized the field of positive-strand RNA viruses, whose genomes have the same polarity as cellular mRNA. The cDNA-based reverse genetics system is a seminal method that enables direct manipulation of the viral genomic RNA, thereby generating recombinant viruses for molecular and genetic studies of both viral RNA elements and gene products in viral replication and pathogenesis. It also provides a valuable platform that allows the development of genetically defined vaccines and viral vectors for the delivery of foreign genes. For many positive-strand RNA viruses such as Japanese encephalitis virus (JEV), however, the cloned cDNAs are unstable, posing a major obstacle to the construction and propagation of the functional cDNA. Here, the present report describes the strategic considerations in creating and amplifying a genetically stable full-length infectious JEV cDNA as a bacterial artificial chromosome (BAC) using the following general experimental procedures: viral RNA isolation, cDNA synthesis, cDNA subcloning and modification, assembly of a full-length cDNA, cDNA linearization, in vitro RNA synthesis, and virus recovery. This protocol provides a general methodology applicable to cloning full-length cDNA for a range of positive-strand RNA viruses, particularly those with a genome of &gt;10 kb in length, into a BAC vector, from which infectious RNAs can be transcribed in vitro with a bacteriophage RNA polymerase.

Here, a protocol is presented for creating an infectious bacterial artificial chromosome containing a full-length cDNA of the positive-strand genomic RNA of Japanese encephalitis virus. This protocol can be used to construct a functional cDNA of other positive-strand RNA viruses, making it a powerful genomic tool for studying virus biology.

Reverse genetics, an approach to rescue infectious virus entirely from a cloned cDNA, has revolutionized the field of positive-strand RNA viruses, whose ...

A Simple and Efficient Approach to Construct Mutant Vaccinia Virus Vectors

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been successfully employed for editing genomes of various organisms. Here we describe a protocol for editing the vaccinia virus (VV) genome in the cytoplasm of VV-infected CV-1 cells using the RNA-guided Cas9. RNA-guided Cas9 induces double-stranded DNA breaks facilitating homologous recombination efficiently and specifically in the targeted site of VV and a transgene can be incorporated into these sites by homologous recombination. By using a site-specific homologous vector with transgene(s), a N1L gene-deleted VV with the red fluorescence protein (RFP) gene incorporated in this region was generated with a successful recombination efficiency 10 times greater than that obtained from the conventional homologous recombination method. This protocol demonstrates successful use of RNA-guided Cas9 system to generate mutant VVs with enhanced efficiency.

Vaccinia virus (VV) has been widely used in biomedical research and the improvement of human health. This article describes a simple, highly efficient method to edit the VV genome using a CRISPR-Cas9 system.

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been ...

Generating Recombinant Avian Herpesvirus Vectors with CRISPR/Cas9 Gene Editing

Herpesvirus of turkeys (HVT) is an ideal viral vector for the generation of recombinant vaccines against a number of avian diseases, such as avian influenza (AI), Newcastle disease (ND), and infectious bursal disease (IBD), using bacterial artificial chromosome (BAC) mutagenesis or conventional recombination methods. The clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 system has been successfully used in many settings for gene editing, including the manipulation of several large DNA virus genomes. We have developed a rapid and efficient CRISPR/Cas9-mediated genome editing pipeline to generate recombinant HVT. To maximize the potential use of this method, we present here detailed information about the methodology of generating recombinant HVT expressing the VP2 protein of IBDV. The VP2 expression cassette is inserted into the HVT genome via an NHEJ (nonhomologous end-joining)-dependent repair pathway. A green fluorescence protein (GFP) expression cassette is first attached to the insert for easy visualization and then removed via the Cre-LoxP system. This approach offers an efficient way to introduce other viral antigens into the HVT genome for the rapid development of recombinant vaccines.

Herpesvirus of turkeys (HVT) is widely used as a vector platform for the generation of recombinant vaccines against a number of avian diseases. This article describes a simple and rapid approach for the generation of recombinant HVT-vectored vaccines using an integrated NHEJ-CRISPR/Cas9 and Cre-Lox system.

Herpesvirus of turkeys (HVT) is an ideal viral vector for the generation of recombinant vaccines against a number of avian diseases, such as avian ...

Generation, Amplification, and Titration of Recombinant Respiratory Syncytial Viruses

The use of recombinant viruses has become crucial in basic or applied virology. Reverse genetics has been proven to be an extremely powerful technology, both to decipher viral replication mechanisms and to study antivirals or provide development platform for vaccines. The construction and manipulation of a reverse genetic system for a negative-strand RNA virus such as a respiratory syncytial virus (RSV), however, remains delicate and requires special know-how. The RSV genome is a single-strand, negative-sense RNA of about 15 kb that serves as a template for both viral RNA replication and transcription. Our reverse genetics system uses a cDNA copy of the human RSV long strain genome (HRSV). This cDNA, as well as cDNAs encoding viral proteins of the polymerase complex (L, P, N, and M2-1), are placed in individual expression vectors under T7 polymerase control sequences. The transfection of these elements in BSR-T7/5 cells, which stably express T7 polymerase, allows the cytoplasmic replication and transcription of the recombinant RSV, giving rise to genetically modified virions. A new RSV, which is present at the cell surface and in the culture supernatant of BSRT7/5, is gathered to infect human HEp-2 cells for viral amplification. Two or three rounds of amplification are needed to obtain viral stocks containing 1 x 106 to 1 x 107 plaque-forming units (PFU)/mL. Methods for the optimal harvesting, freezing, and titration of viral stocks are described here in detail. We illustrate the protocol presented here by creating two recombinant viruses respectively expressing free green fluorescent protein (GFP) (RSV-GFP) or viral M2-1 fused to GFP (RSV-M2-1-GFP). We show how to use RSV-GFP to quantify RSV replication and the RSV-M2-1-GFP to visualize viral structures, as well as viral protein dynamics in live cells, by using video microscopy techniques.

We describe a method for generating and amplifying genetically modified respiratory syncytial viruses (RSVs) and an optimized plaque assay for RSVs. We illustrate this protocol by creating two recombinant viruses that respectively allow quantification of RSV replication and live analysis of RSV inclusion bodies and inclusion bodies-associated granules dynamics.

The use of recombinant viruses has become crucial in basic or applied virology. Reverse genetics has been proven to be an extremely powerful technology, ...

Simplified Reverse Genetics Method to Recover Recombinant Rotaviruses Expressing Reporter Proteins

Rotaviruses are a large and evolving population of segmented double-stranded RNA viruses that cause severe gastroenteritis in the young of many mammalian and avian host species, including humans. With the recent advent of rotavirus reverse genetics systems, it has become possible to use directed mutagenesis to explore rotavirus biology, modify and optimize existing rotavirus vaccines, and develop rotavirus multitarget vaccine vectors. In this report, we describe a simplified reverse genetics system that allows the efficient and reliable recovery of recombinant rotaviruses. The system is based on co-transfection of T7 transcription vectors expressing full-length rotavirus (+)RNAs and a CMV vector encoding an RNA capping enzyme into BHK cells constitutively producing T7 RNA polymerase (BHK-T7). Recombinant rotaviruses are amplified by overseeding the transfected BHK-T7 cells with MA104 cells, a monkey kidney cell line that is highly permissive for virus growth. In this report, we also describe an approach for generating recombinant rotaviruses that express a separate fluorescent reporter protein through the introduction of a 2A translational stop-restart element into genome segment 7 (NSP3). This approach avoids deleting or modifying any of the viral open reading frames, thus allowing the production of recombinant rotaviruses that retain fully functional viral proteins while expressing a fluorescent protein.

Generation of recombinant rotaviruses from plasmid DNA provides an essential tool for the study of rotavirus replication and pathogenesis, and the development of rotavirus expression vectors and vaccines. Herein, we describe a simplified reverse genetics approach for generating recombinant rotaviruses, including strains expressing fluorescent reporter proteins.

Rotaviruses are a large and evolving population of segmented double-stranded RNA viruses that cause severe gastroenteritis in the young of many mammalian ...

Rapid, Seamless Generation of Recombinant Poxviruses using Host Range and Visual Selection

Vaccinia virus (VACV) was instrumental in eradicating variola virus (VARV), the causative agent of smallpox, from nature. Since its first use as a vaccine, VACV has been developed as a vector for therapeutic vaccines and as an oncolytic virus. These applications take advantage of VACV&#8217;s easily manipulated genome and broad host range as an outstanding platform to generate recombinant viruses with a variety of therapeutic applications. Several methods have been developed to generate recombinant VACV, including marker selection methods and transient dominant selection. Here, we present a refinement of a host range selection method coupled with visual identification of recombinant viruses. Our method takes advantage of selective pressure generated by the host antiviral protein kinase R (PKR) coupled with a fluorescent fusion gene expressing mCherry-tagged E3L, one of two VACV PKR antagonists. The cassette, including the gene of interest and the mCherry-E3L fusion is flanked by sequences derived from the VACV genome. Between the gene of interest and mCherry-E3L is a smaller region that is identical to the first ~150 nucleotides of the 3&#8217; arm, to promote homologous recombination and loss of the mCherry-E3L gene after selection. We demonstrate that this method permits efficient, seamless generation of rVACV in a variety of cell types without requiring drug selection or extensive screening for mutant viruses.

This is a method to generate &#8220;scarless&#8221; recombinant vaccinia viruses using host-range selection and visual identification of recombinant viruses.

Vaccinia virus (VACV) was instrumental in eradicating variola virus (VARV), the causative agent of smallpox, from nature. Since its first use as a ...

Subcloning Plus Insertion (SPI) - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors

Gene targeting refers to the precise modification of a genetic locus using homologous recombination. The generation of novel cell lines and transgenic mouse models using this method necessitates the construction of a &#8216;targeting&#8217; vector, which contains homologous DNA sequences to the target gene, and has for many years been a limiting step in the process. Vector construction can be performed in vivo in Escherichia coli cells using homologous recombination mediated by phage recombinases using a technique termed recombineering. Recombineering is the preferred technique to subclone the long homology sequences (>4kb) and various targeting elements including selection markers that are required to mediate efficient allelic exchange between a targeting vector and its cognate genomic locus. Typical recombineering protocols follow an iterative scheme of step-wise integration of the targeting elements and require intermediate purification and transformation steps. Here, we present a novel recombineering methodology of vector assembly using a multiplex approach. Plasmid gap repair is performed by the simultaneous capture of genomic sequence from mouse Bacterial Artificial Chromosome libraries and the insertion of dual bacterial and mammalian selection markers. This subcloning plus insertion method is highly efficient and yields a majority of correct recombinants. We present data for the construction of different types of conditional gene knockout, or knock-in, vectors and BAC reporter vectors that have been constructed using this method. SPI vector construction greatly extends the repertoire of the recombineering toolbox and provides a simple, rapid and cost-effective method of constructing these highly complex vectors.

Subcloning Plus Insertion SPI - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors

Gene targeting methodologies can be used to generate transgenic mice with knockout, knock-in and tagged alleles. Here, we describe an improved method of recombineering in E. coli, that we term &#8216;subcloning plus insertion&#8217;, which can be used to generate custom gene targeting vectors rapidly.

Gene targeting refers to the precise modification of a genetic locus using homologous recombination. The generation of novel cell lines and transgenic ...