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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#160; Co., Ltd</td>
			<td>FRESCO17</td>
			<td></td>
		</tr>
		<tr>
			<td>Clean Bench</td>
			<td>Sujing Antai Air Technology&#160; Co., Ltd</td>
			<td>VD-650-U</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA Electrophoresis Equipment</td>
			<td>Cleaver Scientific&#160; 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&#160; Co., Ltd</td>
			<td>FD0303</td>
			<td></td>
		</tr>
		<tr>
			<td>FastDigest HindIII</td>
			<td>Thermo Fisher Scientific&#160; Co., Ltd</td>
			<td>FD0504</td>
			<td></td>
		</tr>
		<tr>
			<td>FastDigest NheI</td>
			<td>Thermo Fisher Scientific&#160; Co., Ltd</td>
			<td>FD0974</td>
			<td></td>
		</tr>
		<tr>
			<td>FastDigest NotI</td>
			<td>Thermo Fisher Scientific&#160; 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&#160; 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&#160; Co., Ltd</td>
			<td>&#8212;</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>&#8212;</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&#160; 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&#160; 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

Research

JoVE Journal

Biology

466 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

Vaccinia Virus Infection &amp; Temporal Analysis of Virus Gene Expression: Part 1

The family Poxviridae consists of large double-stranded DNA containing viruses that replicate exclusively in the cytoplasm of infected cells. Members of the orthopox genus include variola, the causative agent of human small pox, monkeypox, and vaccinia (VAC), the prototypic member of the virus family. Within the relatively large (~ 200 kb) vaccinia genome, three classes of genes are encoded: early, intermediate, and late. While all three classes are transcribed by virally-encoded RNA polymerases, each class serves a different function in the life cycle of the virus. Poxviruses utilize multiple strategies for modulation of the host cellular environment during infection. In order to understand regulation of both host and virus gene expression, we have utilized genome-wide approaches to analyze transcript abundance from both virus and host cells. Here, we demonstrate time course infections of HeLa cells with Vaccinia virus and sampling RNA at several time points post-infection. Both host and viral total RNA is isolated and amplified for hybridization to microarrays for analysis of gene expression.

Vaccinia Virus Infection & Temporal Analysis of Virus Gene Expression: Part 1

Protocol for Vaccinia infection of HeLa cells and analysis of host and viral gene expression. Part 1 of 3.

The family Poxviridae consists of large double-stranded DNA containing viruses that replicate exclusively in the cytoplasm of infected cells.  Members of ...

Vaccinia Virus Infection &amp; Temporal Analysis of Virus Gene Expression: Part 2

The family Poxviridae consists of large double-stranded DNA containing viruses that replicate exclusively in the cytoplasm of infected cells. Members of the orthopox genus include variola, the causative agent of human small pox, monkeypox, and vaccinia (VAC), the prototypic member of the virus family. Within the relatively large (~ 200 kb) vaccinia genome, three classes of genes are encoded: early, intermediate, and late. While all three classes are transcribed by virally-encoded RNA polymerases, each class serves a different function in the life cycle of the virus. Poxviruses utilize multiple strategies for modulation of the host cellular environment during infection. In order to understand regulation of both host and virus gene expression, we have utilized genome-wide approaches to analyze transcript abundance from both virus and host cells. Here, we demonstrate time course infections of HeLa cells with Vaccinia virus and sampling RNA at several time points post-infection. Both host and viral total RNA is isolated and amplified for hybridization to microarrays for analysis of gene expression.

Vaccinia Virus Infection & Temporal Analysis of Virus Gene Expression: Part 2

Protocol for Vaccinia infection of HeLa cells and analysis of host and viral gene expression. Part 2 of 3.

Vaccinia Virus Infection &amp; Temporal Analysis of Virus Gene Expression: Part 3

Vaccinia Virus Infection & Temporal Analysis of Virus Gene Expression: Part 3

Protocol for Vaccinia infection of HeLa cells and analysis of host and viral gene expression. Part 3 describes the process of fluorescently labeling the amplified RNA from both host and viral samples by amino allyl coupling of dyes. Part 3 of 3.

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks.  These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology.  Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages.  To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis.  The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa).  The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells.  We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

This procedure describes a quick and easy workflow to introduce siRNA into difficult to transfect cell lines and follow gene expression by real-time PCR. Use of an automated cell counter, multi-well electroporation plate, and automated electrophoresis station provide quick and reliable results without the need for expensive robotic handling.

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

A High-content Imaging Workflow to Study Grb2 Signaling Complexes by Expression Cloning

Signal transduction by growth factor receptors is essential for cells to maintain proliferation and differentiation and requires tight control. Signal transduction is initiated by binding of an external ligand to a transmembrane receptor and activation of downstream signaling cascades. A key regulator of mitogenic signaling is Grb2, a modular protein composed of an internal SH2 (Src Homology 2) domain flanked by two SH3 domains that lacks enzymatic activity. Grb2 is constitutively associated with the GTPase Son-Of-Sevenless (SOS) via its N-terminal SH3 domain. The SH2 domain of Grb2 binds to growth factor receptors at phosphorylated tyrosine residues thus coupling receptor activation to the SOS-Ras-MAP kinase signaling cascade. In addition, other roles for Grb2 as a positive or negative regulator of signaling and receptor endocytosis have been described. The modular composition of Grb2 suggests that it can dock to a variety of receptors and transduce signals along a multitude of different pathways1-3.
	Described here is a simple microscopy assay that monitors recruitment of Grb2 to the plasma membrane. It is adapted from an assay that measures changes in sub-cellular localization of green-fluorescent protein (GFP)-tagged Grb2 in response to a stimulus4-6. Plasma membrane receptors that bind Grb2 such as activated Epidermal Growth Factor Receptor (EGFR) recruit GFP-Grb2 to the plasma membrane upon cDNA expression and subsequently relocate to endosomal compartments in the cell. In order to identify in vivo protein complexes of Grb2, this technique can be used to perform a genome-wide high-content screen based on changes in Grb2 sub-cellular localization. The preparation of cDNA expression clones, transfection and image acquisition are described in detail below. Compared to other genomic methods used to identify protein interaction partners, such as yeast-two-hybrid, this technique allows the visualization of protein complexes in mammalian cells at the sub-cellular site of interaction by a simple microscopy-based assay. Hence both qualitative features, such as patterns of localization can be assessed, as well as the quantitative strength of the interaction.

A high-content screening method for the identification of novel signaling competent transmembrane receptors is described. This method is amenable to large-scale automation and allows predictions about in vivo protein binding and the sub-cellular localization of protein complexes in mammalian cells.

Signal  transduction by growth factor receptors is essential for cells to maintain  proliferation and differentiation and requires tight control. Signal  ...

Recombinant Protein Expression for Structural Biology in HEK 293F Suspension Cells: A Novel and Accessible Approach

The expression and purification of large amounts of recombinant protein complexes is an essential requirement for structural biology studies. For over two decades, prokaryotic expression systems such as E. coli have dominated the scientific literature over costly and less efficient eukaryotic cell lines. Despite the clear advantage in terms of yields and costs of expressing recombinant proteins in bacteria, the absence of specific co-factors, chaperones and post-translational modifications may cause loss of function, mis-folding and can disrupt protein-protein interactions of certain eukaryotic multi-subunit complexes, surface receptors and secreted proteins. The use of mammalian cell expression systems can address these drawbacks since they provide a eukaryotic expression environment. However, low protein yields and high costs of such methods have until recently limited their use for structural biology. Here we describe a simple and accessible method for expressing and purifying milligram quantities of protein by performing transient transfections of suspension grown HEK (Human Embryonic Kidney) 293F cells.

The expression of recombinant proteins by mammalian systems is becoming an attractive method for producing protein complexes for structural biology. Here we present a simple yet highly efficient expression system using suspension grown mammalian cells to purify protein complexes for structural studies.

The expression and purification of large amounts of recombinant protein complexes is an essential requirement for structural biology studies. For over two ...

Highly Efficient Ligation of Small RNA Molecules for MicroRNA Quantitation by High-Throughput Sequencing

MiRNA cloning and high-throughput sequencing, termed miR-Seq, stands alone as a transcriptome-wide approach to quantify miRNAs with single nucleotide resolution. This technique captures miRNAs by attaching 3&#8217; and 5&#8217; oligonucleotide adapters to miRNA molecules and allows de novo miRNA discovery. Coupling with powerful next-generation sequencing platforms, miR-Seq has been instrumental in the study of miRNA biology. However, significant biases introduced by oligonucleotide ligation steps have prevented miR-Seq from being employed as an accurate quantitation tool. Previous studies demonstrate that biases in current miR-Seq methods often lead to inaccurate miRNA quantification with errors up to 1,000-fold for some miRNAs1,2. To resolve these biases imparted by RNA ligation, we have developed a small RNA ligation method that results in ligation efficiencies of over 95% for both 3&#8217; and 5&#8242; ligation steps. Benchmarking this improved library construction method using equimolar or differentially mixed synthetic miRNAs, consistently yields reads numbers with less than two-fold deviation from the expected value. Furthermore, this high-efficiency miR-Seq method permits accurate genome-wide miRNA profiling from in vivo total RNA samples2.

MicroRNAs (miRNAs) are a widely conserved class of regulatory molecules. Here we describe a miRNA cloning method that relies upon two potent ligation steps followed by high-throughput sequencing. Our method permits accurate genome-wide quantitation of miRNAs.

MiRNA cloning and high-throughput sequencing, termed miR-Seq, stands alone as a transcriptome-wide approach to quantify miRNAs with single nucleotide ...

Detection of Intracellular Gene Expression in Live Cells of Murine, Human and Porcine Origin Using Fluorescence-labeled Nanoparticles

The reprogramming of somatic cells to induced pluripotent stem cells (iPS) has successfully been performed in different mammalian species including mouse, rat, human, pig and others. The verification of iPS clones mainly relies on the detection of the endogenous expression of different pluripotency genes. These genes mostly represent transcription factors which are located in the cell nucleus. Traditionally, the proof of their endogenous expression is supplied by immunohistochemical staining after fixation of the cells. This approach requires replicate cultures of each clone at this early stage to preserve validated clones for further experiments. The present protocol describes an approach with gene-specific nanoparticles which allows the evaluation of intracellular gene expression directly in live cells by fluorescence. The nanoparticles consist of a central gold particle coupled to a capture strand carrying a sequence complementary to the target mRNA as well as a quenched reporter strand. These nanoparticles are actively endocytosed and the target mRNA displaces the reporter strand which then start to fluoresce. Therefore, specific target gene expression can be detected directly under the microscope. In addition, the emitted fluorescence allows the identification, isolation and enrichment of cells expressing a specific gene by flow cytometry. This method can be applied directly to live cells in culture without any manipulation of the target cells.

This manuscript describes a novel technique which allows the detection of intracellular gene expression after endocytosis of fluorescence-labeled nanoparticles directly in live cells. The method does not require manipulation of the cells and is not restricted with respect to the target gene or species.

The reprogramming of somatic cells to induced pluripotent stem cells (iPS) has successfully been performed in different mammalian species including mouse, ...

High-throughput CRISPR Vector Construction and Characterization of DNA Modifications by Generation of Tomato Hairy Roots

Targeted DNA mutations generated by vectors with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology have proven useful for functional genomics studies. While most cloning strategies are simple to perform, they generally use multiple steps and can require several days to generate the ultimate constructs. The method presented here is based on DNA assembly and can produce fully functional CRISPR vectors in a single cloning reaction. Vector construction can also be pooled, further increasing the efficiency and utility of the process. A modification of the method is used to create CRISPR vectors with multiple gene targets. CRISPR vectors are then transformed into tomato hairy roots to generate transgenic materials with targeted DNA modifications. Hairy roots are a useful system for testing vector functionality as they are technically simple to generate and amenable to large-scale production. The methods presented here will have wide application as they can be used to generate a variety of CRISPR vectors and be used in a wide range of plant species.

Using DNA assembly, multiple CRISPR vectors can be constructed in parallel in a single cloning reaction, making the construction of large numbers of CRISPR vectors a simple task. Tomato hairy roots are an excellent model system to validate CRISPR vectors and generate mutant materials.

Targeted DNA mutations generated by vectors with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology have proven useful for ...

Expression and Purification of Virus-like Particles for Vaccination

Virus-like particles (VLPs) and subviral particles (SVPs) are an alternative approach to viral vaccine design that offers the advantages of increased biosafety and stability over use of live pathogens. Non-infectious and self-assembling, VLPs are used to present structural proteins as immunogens, bypassing the need for live pathogens or recombinant viral vectors for antigen delivery. In this article, we demonstrate the different stages of VLP design and development for future applications in preclinical animal testing. The procedure includes the following stages: selection of antigen, expression of antigen in cell line of choice, purification of VLPs/SVPs, and quantification for antigen dosing. We demonstrate use of both mammalian and insect cell lines for expression of our antigens and demonstrate how methodologies differ in yield. The methodology presented may apply to a variety of pathogens and can be achieved by substituting the antigens with immunogenic structural proteins of the user&#39;s microorganism of interest. VLPs and SVPs assist with antigen characterization and selection of the best vaccine candidates.

Here, we present a protocol for synthesizing virus-like particles using either baculovirus or mammalian expression systems, and ultracentrifugation purification. This highly customizable approach is used to identify viral antigens as vaccine targets in a safe and flexible manner.

Virus-like particles (VLPs) and subviral particles (SVPs) are an alternative approach to viral vaccine design that offers the advantages of increased ...