This work was supported by Consejo Nacional de Ciencia y Tecnolog&#237;a, CONACYT, M&#233;xico (grant number 154833) and Universidad Nacional Aut&#243;noma de M&#233;xico. The authors wish to thank V&#237;ctor Gonz&#225;lez, Rosa I. Santamar&#237;a, Patricia Bustos, and Soledad Ju&#225;rez for their administrative and technical advice.

1. CAPRRESI Protocol
NOTE: Figure 1 represents the overall CAPRRESI protocol. This technique is based on an in silico design and the subsequent construction of the desired chimeras.
<ol>
	<li>Selection of the cloning plasmid, pUC18 or pUC19.
	<ol>
		<li>Select the pUC vector that best fits technical demands. 
		NOTE: Both vectors have the same sequence, except for the orientation of the multiple cloning site. This is important for the design of oligonucleotides and the PCR plasmid disruption.</li>
	</ol>
	</li>
	<li>In silico analysis of the wildtype genes and pUC18/19 sequences.
	<ol>
		<li>Obtain the DNA sequences of the wildtype genes and save them in a FASTA format file (e.g., genes.fas). 
		NOTE: The sequences of pUC18/19 vectors are contained in the pUC.fas file (Figure 1, step 1).</li>
		<li>Determine which restriction enzymes do not cut the wildtype genes and pUC18/19 sequences by running the REsearch.pl script. Alternatively, perform a restriction enzyme site search with an online tool (Figure 1, step 2).
		<ol>
			<li>Type the following into a terminal: 
			perl REsearch.pl genes.fas 
			perl REsearch.pl pUC.fas 
			NOTE: These commands will create the following output files: genes_lin.fas, genes_re.fas, pUC_lin.fas, and pUC_re.fas.</li>
			<li>Select the appropriate restriction enzymes for cloning the wildtype genes into the multiple cloning site of the chosen pUC18/19 vector by comparing the genes_re.fas and pUC.fas files. Choose the orientation of the insert relative to the ampicillin resistance gene (ampR) of the pUC18/19 vector.</li>
		</ol>
		</li>
		<li>Design forward and reverse oligonucleotides to amplify the coding region of the wildtype genes (external oligonucleotides). Include the proper restriction enzyme site at each 5&#39; end of the oligonucleotides; this will define the insert orientation.
		<ol>
			<li>Include a functional ribosome binding site in the forward oligonucleotides.</li>
			<li>Insert both wildtype genes in the same orientation inside the vector.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Selection of the breaking regions for chimera assembly and plasmid disruption.
	<ol>
		<li>Obtain the amino acid sequence of the wildtype and the ampR genes by running the translate.pl script (Figure 1, step 3).
		<ol>
			<li>Type the following into a terminal: 
			perl translate.pl genes.fas 
			perl translate.pl ampR.fas 
			NOTE: This script will create the following output files: genes_aa.fas and ampR_aa.fas.</li>
		</ol>
		</li>
		<li>Globally align the two wildtype amino acid sequences with an appropriate software (e.g., MUSCLE)9.</li>
		<li>Select the desired break regions (3-6 amino acids) for chimera assembly based on the sequence alignment. Save them into a file in FASTA format (e.g., regions.fas).</li>
		<li>Locate the DNA sequences that code for the selected amino acid stretches on both wildtype genes.</li>
	</ol>
	</li>
	<li>In silico generation of synonym DNA sequences containing restriction enzyme sites.
	<ol>
		<li>Obtain all the synonym DNA sequences that code for each amino acid sequence stretch selected as breaking regions (Figure 1, step 4); these sequences were stored in the regions.fas file.
		<ol>
			<li>Type the following into a terminal: 
			perl synonym.pl regions.fas 
			NOTE: This script will create the regions_syn.fas output file.</li>
		</ol>
		</li>
		<li>Search for restriction enzyme sites found on synonym DNA sequences.
		<ol>
			<li>Type the following into a terminal: perl REsynonym.pl regions_syn.fas 
			NOTE: This script will create the regions_syn-re.fas output file.</li>
		</ol>
		</li>
		<li>Choose one restriction enzyme that is shared between synonym sequences of both wildtype genes; this site will be used to assemble chimeras via restriction enzyme digestion. Verify that the chosen restriction enzyme cut neither the wildtype genes nor the pUC18/19 vector sequences.
		<ol>
			<li>Compare the restriction enzymes found on the genes_re.fas, pUC_re.fas, and regions_syn-re.fas files.</li>
		</ol>
		</li>
		<li>In silico substitute the originals for their synonym DNA sequences at the corresponding loci on both wildtype genes. Append synonym DNA sequences into the wildtype sequence file (genes.fas).</li>
		<li>Get the amino acid sequence of genes contained on the genes.fas file (perl translate.pl genes.fas).</li>
		<li>Align the amino acid sequences translated from wildtype and synonym DNA sequences. Verify that both sequences are the same.</li>
		<li>Repeat all the steps of this section with the ampR gene present on the pUC18/19 vector. 
		NOTE: The goal is to disrupt the ampR gene (file ampR.fas) into two complementary parts. The restriction enzyme selected to disrupt the ampR gene should be different from the one used for chimera assembly.</li>
	</ol>
	</li>
	<li>Independent cloning of the two wildtype genes into the selected pUC18/19 vector (Figure 1, step 3).
	<ol>
		<li>Purify the chosen pUC18/19 plasmid DNA using a plasmid DNA purification kit.
		<ol>
			<li>For example, transform E. coli DH5&#945; with pUC18 plasmid and grow the transformants overnight at 37 &#176;C on solid LB-0.3 mg/mL ampicillin (Amp). Pick a transformant colony, inoculate a new LB-0.3 mg/mL Amp plate, and grow it overnight at 37 &#176;C.</li>
			<li>Take part of the previous culture and grow it in liquid LB-0.3 mg/mL Amp for 6-8 h at 37 &#176;C. Extract the plasmid DNA using a kit.</li>
		</ol>
		</li>
		<li>PCR amplify wildtype genes from the total genomic DNA using the appropriate external oligonucleotides. Use a high-fidelity DNA polymerase if possible. Select the DNA polymerase that maximizes the yield. Perform PCR according to the manufacturer&#39;s guidelines and the melting temperature of the oligonucleotides.
		<ol>
			<li>Run PCR cycles, depending upon the DNA polymerase, amplicon size, template, and oligonucleotides used. For example, to amplify rpoD DNA, prepare a 50 &#181;L reaction: 5 &#181;L of 10x buffer, 2 &#181;L of 50 mM MgSO4, 1 &#181;L of 10 mM dNTP mix, 2 &#181;L of each 10-&#181;M oligonucleotide (Table 2), 2 &#181;L of template DNA (E. coli DH5&#945; total DNA), 35.8 &#181;L of ultra-pure water, and 0.2 &#181;L of high-fidelity DNA polymerase. Run the following PCR cycles: 1 min at 94 &#176;C for the initial denaturation followed by 30 cycles of amplification (30 s at 94 &#176;C to denature, 30 s at 60 &#176;C to anneal the oligonucleotides, and 2 min at 68 &#176;C to extend).</li>
			<li>Dissolve 1.2 g of agarose in 100 mL of double-distilled water by heating them in the microwave. Assemble a gel tray and comb and put them into the horizontal gel caster. Fill the gel tray with the melted agarose solution and let it solidify. Remove the comb.</li>
			<li>Place the gel into the electrophoresis chamber. Fill chamber with 1x Tris-acetate-EDTA buffer. Load 50 &#181;L of PCR products into the gel wells. Electrophorese the samples (e.g., at 110 V for 1 h).</li>
			<li>After electrophoresis, stain the gel in 100 mL of double-distilled water containing 100 &#181;L of 1 mg/mL ethidium bromide solution for 10 min with slow shaking. Wash the gel in double-distilled water for another 10 min in slow shaking; use a horizontal shaker. 
			Caution: Ethidium bromide is a toxic compound; wear protective gloves and a cotton laboratory coat.</li>
			<li>Purify wildtype gene DNA from the corresponding bands of the gel using a kit. Visualize the bands by placing the stained gel in a UV chamber (recommended wavelength: 300 nm). Keep the exposure of the gel to a minimum. Use a DNA purification kit and follow the manufacturer&#39;s instructions. 
			Caution: UV radiation is dangerous; wear a protective shield, glasses, and a cotton laboratory coat.</li>
		</ol>
		</li>
		<li>Digest purified wildtype genes and pUC18/19 plasmid DNA with the restriction enzymes according to the manufacturer&#39;s instructions. Use fast digestion enzymes when possible. For example, digest 6 &#181;L of pUC18 DNA (300 ng/&#181;L) in 10 &#181;L of ultrapure water, 2 &#181;L of 10X buffer, 1 &#181;L of KpnI restriction enzyme, and 1 &#181;L of XbaI restriction enzyme. Leave the reaction at 37 &#176;C for 30 min.
		<ol>
			<li>Inactivate the restriction enzymes according to the manufacturer&#39;s guidelines. For example, inactivate the double-digestion reaction KpnI-XbaI at 80 &#176;C for 5 min.</li>
		</ol>
		</li>
		<li>Mix insert:vector DNA at a volumetric ratio of 3:1. Follow the manufacturer&#39;s instructions for a ligation reaction. Use fast ligation enzymes when possible (leave the reaction at 25 &#176;C for 10 min). 
		NOTE: Typically, the DNA concentration after purification using the kits is good enough for digestion and ligation reactions. For example, add 6 &#181;L of rpoD DNA (KpnI-XbaI, 150 ng/&#181;L), 3 &#181;L of pUC18 DNA (KpnI-XbaI, 150 ng/&#181;L), 10 &#181;L of 2x buffer, 1 &#181;L of ultrapure water, and 1 &#181;L of T4 DNA ligase. Leave ligation reaction at 25 &#176;C for 10 min.</li>
		<li>Transform the E. coli DH5&#945; with 2-4 &#181;L of the ligation reaction, as described in the Supplementary Materials. Plate the transformed cells into solid LB/Amp/X-gal/IPTG plates. Leave the plates overnight at 37 &#176;C.</li>
		<li>Select white-colored transformant E. coli colonies and streak them on a new LB/Amp plate. Leave the plates overnight at 37 &#176;C.</li>
		<li>Perform a colony PCR to choose candidates for sequencing the reaction, as described in the Supplementary Materials. Extract plasmid DNA from candidates. Alternatively, digest candidate plasmid DNA with the same restriction enzymes used for cloning the inserts.</li>
		<li>Sequence candidate constructions with a sequencing service provider10. Assemble the sequence in silico using a DNA assembler11(Figure 1, step 5).</li>
	</ol>
	</li>
	<li>Plasmid disruption by PCR followed by restriction enzyme digestion.
	<ol>
		<li>Design in silico forward and reverse oligonucleotides at break regions for wildtype and ampR genes, respectively. Substitute in silico the wildtype fragment for its corresponding synonym DNA sequence (obtained in step 1.4). 
		NOTE: This synonym DNA sequence contains a restriction enzyme site. Forward and reverse oligonucleotides overlap at the restriction enzyme site. Oligonucleotides should range from 21-27 nucleotides, end with a cytosine or guanine, and contain a restriction enzyme site. A forward oligonucleotide has the same sequence as its target region on the template DNA. A reverse oligonucleotide is the reverse complementary sequence of the target region on the template DNA.</li>
		<li>Use the two wildtype gene constructions as the DNA template for PCR reactions (e.g., pUC18rpoD and pUC18sigA). Obtain two complementary parts of each construction (pUC18rpoD&#963;1-&#963;2, pUC18rpoD&#963;3-&#963;4, pUC18sigA&#963;1-&#963;2, and pUC18sigA&#963;3-&#963;4) (Figure 1, step 6).</li>
		<li>Load DNA samples into an agarose gel 1-1.2% [w/v]. Separate the PCR products from the DNA template by electrophoresis (e.g., 110 V for 1 h). Alternatively, digest the PCR reactions with DpnI to break the DNA template. 
		NOTE: DpnI enzyme recognizes only methylated DNA sequences (5&#39;-GATC-3&#39;).
		<ol>
			<li>Purify the samples using a DNA purification kit. Follow the manufacturer&#39;s guidelines.</li>
		</ol>
		</li>
		<li>Double-digest all the complementary DNA fragments with the appropriate restriction enzymes according to the manufacturer&#39;s directions. Use fast-digest restriction enzymes when possible. For example, double-digest the pUC18rpoD&#963;1-&#963;2 DNA fragment with AflII and SpeI using the manufacturer&#39;s protocol. Leave the digestion reaction at 37 &#176;C for 15 min.</li>
		<li>Inactivate the restriction enzymes according to the manufacturer&#39;s guidelines. For example, inactivate AflII-SpeI at 80 &#176;C for 20 min.</li>
	</ol>
	</li>
	<li>Plasmid recovery using DNA ligation and bacterial transformation.
	<ol>
		<li>Mix the proper DNA fragments in a volumetric 1:1 ratio to assemble the desired chimeric gene (Figure 1, step 7). 
		NOTE: In this way, the integrity of the ampR gene is restored, producing a functional protein.
		<ol>
			<li>For example, mix 4 &#181;L of pUC18rpoD&#963;1-&#963;2 DNA (digested with AflII-SpeI, 150 ng/&#181;L), 4 &#181;L of pUC18sigA&#963;3-&#963;4 DNA (AflII-SpeI, 150 ng/&#181;L), 10 &#181;L of 2x buffer, 2 &#181;L of ultrapure water, and 1 &#181;L of T4 DNA ligase; the DNA concentration after kit purification is good enough for digestion and subsequent ligation. Leave the ligation reaction at 25 &#176;C for 10 min.</li>
		</ol>
		</li>
		<li>Transform E. coli DH5&#945; with 3-5 &#181;L of the chimera assembly ligation reaction (Supplementary Materials). Grow the transformant cells in LB/Amp/X-gal/IPTG plates overnight at 37 &#176;C overnight.</li>
		<li>Select white-colored transformant E. coli colonies and streak them on a new LB/Amp plate. Leave the plates overnight at 37 &#176;C.</li>
		<li>Perform a colony PCR to choose candidates for a sequencing reaction, as described in the Supplementary Materials. Visualize bands in a 1-1.2% (w/v) agarose gel by performing electrophoresis (described in step 2.3.2.1).</li>
		<li>Grow cultures from positive candidates. Extract plasmid DNA from them using a plasmid DNA purification kit.</li>
	</ol>
	</li>
</ol>
2. Sequence Candidate Chimeric Constructions
<ol>
	<li>Make sure the quality of the plasmid DNA is good enough for the sequencing reaction by following guidelines from the sequence service provider. Extract DNA using purification kits. For example, on the internet page of the sequence service provider, pay special attention to the recommended DNA concentration for the samples. Obtain the concentration of samples using a DNA quantification instrument.</li>
	<li>Sequence candidate chimeric constructions with a sequencing service provider10.</li>
	<li>Assemble sequencing reads with an in silico DNA assembler11.</li>
	<li>Align assembled versus in silico-designed chimeric sequences using sequence alignment tools9,12. Verify that the chimeric gene was fused successfully.</li>
</ol>
3. Making Preparations
NOTE: All steps involving living cells should be performed in a clean laminar flow hood with the Bunsen burner on.
<ol>
	<li>Producing E. coli DH5&#945;-competent cells.
	<ol>
		<li>To produce E. coli-competent cells, follow published protocols13 or see the Supplementary Materials. Alternatively, use commercially available competent cells.</li>
	</ol>
	</li>
	<li>Transforming E. coli DH5&#945;-competent cells.
	<ol>
		<li>For the chemical transformation of E. coli cultures, follow published protocols13 or see the Supplementary Materials. Alternatively, use electroporation for bacterial transformation. If commercially available competent cells are used, follow the manufacturer&#39;s guidelines.</li>
	</ol>
	</li>
	<li>Purifying genomic and plasmid DNA from E. coli DH5&#945;.
	<ol>
		<li>Perform nucleic acid extraction, as stated in the Supplementary Materials, using commercially available kits or following published protocols13.</li>
	</ol>
	</li>
	<li>Perform colony PCR.
	<ol>
		<li>Use this technique to identify candidate transformant colonies for subsequent sequencing reaction. 
		NOTE: This method does not represent a final verification of the integrity of the sequences. A detailed description of this technique is available in the Supplementary Materials.</li>
	</ol>
	</li>
	<li>Preparing the solutions.
	<ol>
		<li>See Table 1 for more information about solution preparation.</li>
	</ol>
	</li>
	<li>Download the scripts and sequence files required for the CAPRRESI protocol.
	<ol>
		<li>Download gene bank files containing the complete genome sequence of the desired species, extract the DNA sequence of the chosen gene using a genome browser, and save it in fasta file format. Download the Perl scripts required for the CAPRRESI protocol. Store the scripts and the sequence files in the same directory.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Horton, R. M., Hunt, H. D., Ho, S. N., Pullen, J. K., Pease, L. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+hybrid+genes+without+the+use+of+restriction+enzymes:+gene+splicing+by+overlap+extension.">Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension.</a> Gene. 77 (1), 61-68 (1989).</li><li>Vos, M. J., Kampinga, H. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+PCR+amplification+strategy+for+unrestricted+generation+of+chimeric+genes.">A PCR amplification strategy for unrestricted generation of chimeric genes.</a> Anal. Biochem. 380 (2), 338-340 (2008).</li><li>Hawkins, N. C., Garriga, G., Beh, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Creating+Precise+GFP+Fusions+in+Plasmids+Using+Yeast+Homologous+Recombination.">Creating Precise GFP Fusions in Plasmids Using Yeast Homologous Recombination.</a> Biotechniques. 34 (1), 1-5 (2003).</li><li>Sander, J. D., Joung, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPR-Cas+systems+for+editing,+regulating+and+targeting+genomes.">CRISPR-Cas systems for editing, regulating and targeting genomes.</a> Nat Biotechnol. 32 (4), 347-355 (2014).</li><li>Nagy, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cre+recombinase:+The+universal+reagent+for+genome+tailoring.">Cre recombinase: The universal reagent for genome tailoring.</a> Genesis. 26 (2), 99-109 (2000).</li><li>Gibson, D. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzymatic+assembly+of+DNA+molecules+up+to+several+hundred+kilobases.">Enzymatic assembly of DNA molecules up to several hundred kilobases.</a> Nature Methods. 6 (5), 343-345 (2009).</li><li>Norrander, J., Kempe, T., Messing, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Construction+of+improved+M13+vectors+using+oligodeoxynucleotide-directed+mutagenesis.">Construction of improved M13 vectors using oligodeoxynucleotide-directed mutagenesis.</a> Gene. 26 (1), 101-106 (1983).</li><li>Gruber, T. M., Gross, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+sigma+subunits+and+the+partitioning+of+bacterial+transcription+space.">Multiple sigma subunits and the partitioning of bacterial transcription space.</a> Annu Rev Microbiol. 57, 441-466 (2003).</li><li>Edgar, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MUSCLE:+multiple+sequence+alignment+with+high+accuracy+and+high+throughput.">MUSCLE: multiple sequence alignment with high accuracy and high throughput.</a> Nucleic Acids Res. 32 (5), 1792-1797 (2004).</li><li>Thompson, J. D., Gibson, T. J., Higgins, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+sequence+alignment+using+ClustalW+and+ClustalX.">Multiple sequence alignment using ClustalW and ClustalX.</a> Curr Protoc Bioinformatics. , 1-22 (2002).</li><li>Sambrook, J., Russell, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+Cloning:+A+laboratory+manual.">Molecular Cloning: A laboratory manual.</a> CSHLP. , (2001).</li><li>Rutherford, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Artemis:+sequence+visualization+and+annotation.">Artemis: sequence visualization and annotation.</a> Bioinformatics. 16 (10), 944-945 (2000).</li></ol>

The authors declare that they have no competing financial interests.

CAPRRESI was designed as an alternative to the PRM2. The original PRM is a powerful technique; it allows for the fusion of DNA sequences along any part of the selected genes. For PRM, wildtype genes should be cloned into the same plasmid. After that, oligonucleotides are designed inside wildtype and antibiotics resistance genes found on the plasmid. Plasmid disruption is achieved by PCR using blunt-ended, high-fidelity DNA polymerases and previously designed oligonucleotides. The ligation of complementary PCR products assembles chimeric genes and restores the antibiotics resistance cassette, resulting in functional plasmid recovery. PRM enables the construction of chimeric gene libraries on the same plasmid background. Unfortunately, the ligation of blunt-ended DNA sequences could be technically arduous. Chimera assembly by overlapping PCR fragments1 also requires that DNA polymerases yield blunt-ended products and DNA purification for each reaction. The chimeric genes assembled by this technique are linear DNA products. The cloning of each construction into the target vector could delay further analysis.
CAPRRESI benefits from many of the strengths of PRM and also simplifies the ligation of complementary DNA fragments by using restriction enzyme sites on synonym DNA sequence regions. For these reasons, CAPRRESI does not require blunt-ended DNA polymerases. The use of synonym DNA sequences restrains fusion sites for chimera assembly but enhances ligation efficiency. Another important modification introduced in CAPRRESI is that chimeras are assembled and handled in pUC18/19 vectors. These vectors possess several advantageous features, as stated previously. Chimeric DNA could be obtained by propagating the construction vector in E. coli hosts. Subsequent cloning of chimeric genes into the final plasmid (e.g., an expression vector) is sometimes required.
CAPRRESI also relies on the in silico handling of DNA and amino acid sequences. Ad hoc Perl scripts enable the selection of the appropriate restriction enzymes to clone wildtype genes, the calculation of all synonym DNA sequences corresponding to break regions (for chimera assembly), and the identification of restriction enzymes for simplifying plasmid recovery. Given these conditions, CAPRRESI is an alternative for fusing protein-coding genes. Critical steps of the CAPRRESI protocol are: 1) the selection of breaking regions and 2) the purification of PCR products. Breaking regions are stretches where synonym sequences are calculated, producing restriction enzyme sites that are not found on wildtype sequences. The use of restriction enzyme sites for chimera assembly eliminates the need for DNA polymerases that yield blunt-ended products and eases the ligation reaction. Not all regions will have usable restriction enzyme sites; for this reason, it is recommended to move breaking regions by one amino acid at a time until the best sequence stretch is found. If the in silico design does not allow for the movement of the breaking regions or the sequence stretches lack synonym DNA sequences with suitable restriction enzyme sites, it is recommended to fuse target genes using overlapping oligonucleotides and to introduce synonym DNA sequences into the ampicillin resistance gene (found on the vector) only. In this way, the genes can be fused at any part and the plasmid recovery relies on the ligation of one unique site (digested with only one restriction enzyme). The flexibility of CAPPRESI allows for it to be performed in combination with other techniques.
The purification of PCR products is performed to eliminate template DNA, decreasing the chances of parental plasmid contamination after the ligation of complementary DNA fragments. Fulfilling these two critical steps enhances bacterial transformation efficiency (the number of correctly assembled constructions), allowing CAPRRESI to be performed with either chemically competent (CaCl2) or electrocompetent E. coli cells. The first type of competent cells represents the cheapest source of E. coli cultures, employed for bacterial transformation in most laboratories. Because of all the features shown previously, CAPRRESI represents a useful option for assembling chimeras.
Moreover, CAPRRESI could work in combination with overlapping PCR fragments or plasmid recovery techniques. For example, instead of inserting two synonym sequences per complementary DNA portion, the desired breaking region (on the target wildtype genes) could use overlapping oligonucleotides to fuse intervening sequences. The introduction of synonym sequences could be restricted to the ampR gene of pUC18/19 vectors, leading to the use of only one restriction enzyme to restore plasmid integrity. These future applications could strengthen CAPRRESI by allowing for the fusion of any type of DNA sequences (i.e., not only protein-coding genes), without compromising ligation efficiency.
In order to test CAPRRESI, two chimeric sigma factors were assembled by exchanging regions of two wildtype primary sigma factor genes: rpoD (E. coli) and sigA (R. etli). All known primary sigma factors hold four domains, &#963;1 to &#963;48. Chimera 01 consists of RpoD&#963;1-&#963;2 and SigA&#963;3-&#963;4, while chimera 02 has SigA&#963;1-&#963;2 and RpoD&#963;3-&#963;4. The integrity of the chimeric constructions was verified by Sanger sequencing10 (Figure 2).

Chimeric gene assembly has been widely used in molecular biology to elucidate protein function and/or for biotechnological purposes. Different methods exist for fusing genes, such as overlapping PCR product amplification1, plasmid recovery2, homologous recombination3, CRISPR-Cas9 systems4, site-directed recombination5, and Gibson assembly6. Each of these offers different technical advantages; for example, the flexibility of overlapping PCR design, the in vivo selection of constructions during plasmid recovery, or the high efficiency of CRISPR-Cas9 and Gibson systems. On the other hand, some difficulties can arise while performing some of these methods; for example, the first two approaches rely on blunt-ended DNA fragments, and ligation of these types of products could be technically challenging compared to sticky-ended ligation. Site-directed recombination can leave traces of extra DNA sequences (scars) on the original, like in the Cre-loxP system5. CRISPR-Cas9 can sometimes modify other genome regions in addition to the target site4.
Here, we introduce chimera assembly by plasmid recovery and restriction enzyme site insertion (CAPRRESI), a protocol for fusing protein-coding genes that combines the plasmid recovery method (PRM) with the insertion of restriction enzyme sites on synonym DNA sequences, enhancing ligation efficiency. To ensure amino acid sequence integrity, restriction enzyme sites are inserted on synonym DNA sequence stretches. Among the benefits of CAPPRESI are that it can be performed using ordinary laboratory reagents/tools (e.g., enzymes, competent cells, solutions, and thermocycler) and that it can give quick results (when the appropriate enzymes are used). Relying on restriction enzyme sites that emerge from synonym DNA sequences can limit the selection of the exact fusion points inside the proteins of interest. In such cases, target genes should be fused using overlapping oligonucleotides, and restriction enzyme sites should be inserted onto the resistance gene of the vector.
CAPRRESI consists of seven simple steps (Figure 1): 1) selection of the cloning vector, pUC18 or pUC196; 2) in silico analysis of the wildtype sequences to be fused; 3) selection of breaking regions for chimera assembly and plasmid disruption; 4) in silico generation of synonym DNA sequences containing restriction enzyme sites; 5) independent cloning of the wildtype genes into the selected plasmid; 6) plasmid disruption by PCR, followed by restriction enzyme digestion; and 7) plasmid recovery using DNA ligation and bacterial transformation. Chimeric genes produced by this technique should be verified with sequencing.
The pUC18/19 vectors offer technical advantages for cloning and chimera assembly, such as small size (i.e., 2,686 base pairs), high copy number, advantageous sequencing reaction features, and commercial availability7. Here, an Escherichia coli host was used to assemble and handle the chimeras because bacterial cultures are cheap and grow fast. Given this, subsequent cloning of the fusion fragments into the final target plasmids will be needed (e.g., expression vectors as pRK415 in bacteria or pCMV in mammalian cells).
CAPRRESI was tested for fusing two primary sigma factor genes: E. colirpoD and Rhizobium etlisigA. Primary sigma factors are RNA polymerase subunits responsible for transcription initiation, and they consist of four domains (i.e., &#963;1, &#963;2, &#963;3, and &#963;4)8. The amino acid sequence length of proteins encoded by rpoD and sigA are 613 and 685, respectively. RpoD and SigA share 48% identity (98% coverage). These primary sigma factors were split into two complementary fragments between regions &#963;2 and &#963;3. Two chimeric genes were assembled according to this design: chimera 01 (RpoD&#963;1-&#963;2 + SigA&#963;3-&#963;4) and chimera 02 (SigA&#963;1-&#963;2 + RpoD&#963;3-&#963;4). DNA fusion products were verified by sequencing.

<table border="0" cellpadding="0" cellspacing="0" width="756">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Platinum Taq DNA polymerase High Fidelity</td>
			<td style="width:71px;">Thermo Fisher</td>
			<td style="width:115px;">11304011</td>
			<td style="width:355px;">Produces a mix of blunt/3&#8217;-A overhang ended PCR products</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">High pure plasmid isolation kit</td>
			<td style="width:71px;">Roche</td>
			<td style="width:115px;">11754785001</td>
			<td>Used for all plasmid purification reactions</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">High pure PCR product purification kit</td>
			<td style="width:71px;">Roche</td>
			<td style="width:115px;">11732676001</td>
			<td>Used for all PCR purification reactions from agarose gels</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">AflII restriction enzyme</td>
			<td style="width:71px;">New England Biolabs</td>
			<td style="width:115px;">R0520L</td>
			<td>Recognizes sequence 5&#8217;-CTTAAG-3&#8217; and cuts at 37 &#176;C</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">KpnI-HF restriction enzyme</td>
			<td style="width:71px;">New England Biolabs</td>
			<td style="width:115px;">R3142L</td>
			<td>Recognizes sequence 5&#8217;-GGTACC-3&#8217; and cuts at 37 &#176;C</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">SpeI-HF restriction enzyme</td>
			<td style="width:71px;">New England Biolabs</td>
			<td style="width:115px;">R3133L</td>
			<td>Recognizes sequence 5&#8217;-ACTAGT-3&#8217; and cuts at 37 &#176;C</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">XbaI restriction enzyme</td>
			<td style="width:71px;">New England Biolabs</td>
			<td style="width:115px;">R0145L</td>
			<td>Recognizes sequence 5&#8217;-TCTAGA-3&#8217; and cuts at 37 &#176;C</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ampicillin sodium salt</td>
			<td style="width:71px;">Sigma Aldrich</td>
			<td style="width:115px;">A0166-5G</td>
			<td>Antibiotics</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Nalidixic acid</td>
			<td style="width:71px;">Sigma Aldrich</td>
			<td style="width:115px;">N8878-5G</td>
			<td>Antibiotics</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Yeast Extract</td>
			<td style="width:71px;">Sigma Aldrich</td>
			<td style="width:115px;">Y1625-1KG</td>
			<td>Bacterial cell culture</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Casein peptone</td>
			<td style="width:71px;">Sigma Aldrich</td>
			<td style="width:115px;">70171-500G</td>
			<td>Bacterial cell culture</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">NaCl</td>
			<td style="width:71px;">Sigma Aldrich</td>
			<td style="width:115px;">S9888-1KG</td>
			<td>Sodium chloride</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Agar</td>
			<td style="width:71px;">Sigma Aldrich</td>
			<td style="width:115px;">05040-1KG</td>
			<td>Bacterial cell culture</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">XbaI restriction enzyme</td>
			<td style="width:71px;">Thermo Fisher</td>
			<td style="width:115px;">FD0684</td>
			<td>Fast digest XbaI enzyme</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">KpnI restriction enzyme</td>
			<td style="width:71px;">Thermo Fisher</td>
			<td style="width:115px;">FD0524</td>
			<td>Fast digest KpnI enzyme</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Quick Ligation Kit</td>
			<td style="width:71px;">New England Biolabs</td>
			<td style="width:115px;">M2200S</td>
			<td>Fast DNA ligation kit</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">AflII restriction enzyme</td>
			<td style="width:71px;">Thermo Fisher</td>
			<td style="width:115px;">FD0834</td>
			<td>Fast digest AflII enzyme</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">SpeI restriction enzyme</td>
			<td style="width:71px;">Thermo Fisher</td>
			<td style="width:115px;">FD1253</td>
			<td>Fast digest SpeI enzyme</td>
		</tr>
	</tbody>
</table>

caprresi: chimera assembly by plasmid recovery and restriction enzyme site insertion

Here, we present chimera assembly by plasmid recovery and restriction enzyme site insertion (CAPRRESI). CAPRRESI benefits from many strengths of the original plasmid recovery method and introduces restriction enzyme digestion to ease DNA ligation reactions (required for chimera assembly). For this protocol, users clone wildtype genes into the same plasmid (pUC18 or pUC19). After the in silico selection of amino acid sequence regions where chimeras should be assembled, users obtain all the synonym DNA sequences that encode them. Ad hoc Perl scripts enable users to determine all synonym DNA sequences. After this step, another Perl script searches for restriction enzyme sites on all synonym DNA sequences. This in silico analysis is also performed using the ampicillin resistance gene (ampR) found on pUC18/19 plasmids. Users design oligonucleotides inside synonym regions to disrupt wildtype and ampR genes by PCR. After obtaining and purifying complementary DNA fragments, restriction enzyme digestion is accomplished. Chimera assembly is achieved by ligating appropriate complementary DNA fragments. pUC18/19 vectors are selected for CAPRRESI because they offer technical advantages, such as small size (2,686 base pairs), high copy number, advantageous sequencing reaction features, and commercial availability. The usage of restriction enzymes for chimera assembly eliminates the need for DNA polymerases yielding blunt-ended products. CAPRRESI is a fast and low-cost method for fusing protein-coding genes.

Watch this Scientific Journal Video about CAPRRESI: Chimera Assembly by Plasmid Recovery and Restriction Enzyme Site Insertion at JoVE.com

CAPRRESI: Chimera Assembly by Plasmid Recovery and Restriction Enzyme Site Insertion | Biology | Video

CAPRRESI: Chimera Assembly by Plasmid Recovery and Restriction Enzyme Site Insertion

Here, we present chimera assembly by plasmid recovery and restriction enzyme site insertion (CAPRRESI), a protocol based on the insertion of restriction enzyme sites into synonym DNA sequences and functional plasmid recovery. This protocol is a fast and low-cost method for fusing protein-coding genes.

Here, we present chimera assembly by plasmid recovery and restriction enzyme site insertion (CAPRRESI). CAPRRESI benefits from many strengths of the ...

caprresi-chimera-assembly-plasmid-recovery-restriction-enzyme-site

Nanyang Technological University

Research

Education

JoVE Journal

JoVE Core

Biology

Genetics

Biotechnology

SCIENTISTS IN ACTION

11.5K Views.  Universidad Nacional Autónoma de México. Here, we present chimera assembly by plasmid recovery and restriction enzyme site insertion (CAPRRESI), a protocol based on the insertion of restriction enzyme sites into synonym DNA sequences and functional plasmid recovery. This protocol is a fast and low-cost method for fusing protein-coding genes.

Video: CAPRRESI: Chimera Assembly by Plasmid Recovery and Restriction Enzyme Site Insertion

Principles of Site-Specific Recombinase (SSR) Technology

Site-specific recombinase (SSR) technology allows the manipulation of gene structure to explore gene function and has become an integral tool of molecular biology. Site-specific recombinases are proteins that bind to distinct DNA target sequences. The Cre/lox system was first described in bacteriophages during the 1980's. Cre recombinase is a Type I topoisomerase that catalyzes site-specific recombination of DNA between two loxP (locus of X-over P1) sites. The Cre/lox system does not require any cofactors. LoxP sequences contain distinct binding sites for Cre recombinases that surround a directional core sequence where recombination and rearrangement takes place. When cells contain loxP sites and express the Cre recombinase, a recombination event occurs. Double-stranded DNA is cut at both loxP sites by the Cre recombinase, rearranged, and ligated ("scissors and glue"). Products of the recombination event depend on the relative orientation of the asymmetric sequences.
SSR technology is frequently used as a tool to explore gene function. Here the gene of interest is flanked with Cre target sites loxP ("floxed"). Animals are then crossed with animals expressing the Cre recombinase under the control of a tissue-specific promoter. In tissues that express the Cre recombinase it binds to target sequences and excises the floxed gene. Controlled gene deletion allows the investigation of gene function in specific tissues and at distinct time points. Analysis of gene function employing SSR technology --- conditional mutagenesis -- has significant advantages over traditional knock-outs where gene deletion is frequently lethal.

Principles of Site-Specific Recombinase SSR Technology

The advent of site-specific recombinase (SSR) technology and the Cre/lox system has led to numerous advances in molecular biology, and has proven itself as a valuable tool for assessing gene function in transgenic animals. This interview discusses the mechanism of site specific recombination by Cyclization recombinase (Cre) and how the use of this enzyme has led to the development of conditional mutagenesis, which has significant advantages over traditional knock out strategies.

Site-specific recombinase (SSR) technology allows the manipulation of gene structure to explore gene function and has become an integral tool of molecular ...

Assembly, Loading, and Alignment of an Analytical Ultracentrifuge Sample Cell

The analytical ultracentrifuge (AUC) is a powerful biophysical tool that allows us to record macromolecular sedimentation profiles during high speed centrifugation. When properly planned and executed, an AUC sedimentation velocity or sedimentation equilibrium experiment can reveal a great deal about a protein in regards to size and shape, sample purity, sedimentation coefficient, oligomerization states and protein-protein interactions.
This technique, however, requires a rigorous level of technical attention. Sample cells hold a sectored center piece sandwiched between two window assemblies. They are sealed with a torque pressure of around 120-140 in/lbs. Reference buffer and sample are loaded into the centerpiece sectors and then after sealing, the cells are precisely aligned into a titanium rotor so that the optical detection systems scan both sample and reference buffer in the same radial path midline through each centerpiece sector while rotating at speeds of up to 60, 000 rpm and under very high vacuum
Not only is proper sample cell assembly critical, sample cell components are very expensive and must be properly cared for to ensure they are in optimum working condition in order to avoid leaks and breakage during experiments. Handle windows carefully, for even the slightest crack or scratch can lead to breakage in the centrifuge. The contact between centerpiece and windows must be as tight as possible; i.e. no Newton s rings should be visible after torque pressure is applied. Dust, lint, scratches and oils on either the windows or the centerpiece all compromise this contact and can very easily lead to leaking of solutions from one sector to another or leaking out of the centerpiece all together. Not only are precious samples lost, leaking of solutions during an experiment will cause an imbalance of pressure in the cell that often leads to broken windows and centerpieces. In addition, plug gaskets and housing plugs must be securely in place to avoid solutions being pulled out of the centerpiece sector through the loading holes by the high vacuum in the centrifuge chamber. Window liners and gaskets must be free of breaks and cracks that could cause movement resulting in broken windows.
This video will demonstrate our procedures of sample cell assembly, torque, loading and rotor alignment to help minimize component damage, solution leaking and breakage during the perfect AUC experiment.

The analytical ultracentrifuge (AUC) sample cell holds sample and reference buffer and during experiments and is exposed to high vacuum and rotor speeds up to 60,000 rpm. This video will demonstrate the rigorous attention to detail necessary for assembly, loading and alignment of this very important component of an AUC experiment.

The analytical ultracentrifuge (AUC) is a powerful biophysical tool that allows us to record macromolecular sedimentation profiles during high speed ...

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti. 

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the inner ear (fig 1). Hair cells in the developing cochlea, which are the mechanosensory cells of the auditory system, are aligned in one row of inner hair cells and three (in the base and mid-turns) to four (in the apical turn) rows of outer hair cells that span the length of the organ of Corti. Hair cells transduce sound-induced mechanical vibrations of the basilar membrane into neural impulses that the brain can interpret. Most cases of sensorineural hearing loss are caused by death or dysfunction of cochlear hair cells.
An increasingly essential tool in auditory research is the isolation and in vitro culture of the organ explant 1,2,9. Once isolated, the explants may be utilized in several ways to provide information regarding normative, anomalous, or therapeutic physiology. Gene expression, stereocilia motility, cell and molecular biology, as well as biological approaches for hair cell regeneration are examples of experimental applications of organ of Corti explants.
This protocol describes a method for the isolation and culture of the organ of Corti from neonatal mice. The accompanying video includes stepwise directions for the isolation of the temporal bone from mouse pups, and subsequent isolation of the cochlea, spiral ligament, and organ of Corti. Once isolated, the sensory epithelium can be plated and cultured in vitro in its entirety, or as a further dissected micro-isolate that lacks the spiral limbus and spiral ganglion neurons. Using this method, primary explants can be maintained for 7-10 days. As an example of the utility of this procedure, organ of Corti explants will be electroporated with an exogenous DsRed reporter gene. This method provides an improvement over other published methods because it provides reproducible, unambiguous, and stepwise directions for the isolation, microdissection, and primary culture of the organ of Corti.

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti.

This procedure describes a method for the isolation and culture of the murine organ of Corti with or without the spiral limbus and spiral ganglion neurons. We also demonstrate a method for the expression of an exogenous reporter gene in the organ of Corti explant by electroporation.

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the ...

Production of Transgenic Xenopus laevis by Restriction Enzyme Mediated Integration and Nuclear Transplantation

Stable integration of cloned gene products into the Xenopus genome is necessary to control the time and place of expression, to express genes at later stages of embryonic development, and to define how enhancers and promoters regulate gene expression within the embryo. The protocol demonstrated here can be used to efficiently produce transgenic Xenopus laevis embryos. This transgenesis approach involves three parts: 1. Sperm nuclei are isolated from adult X. laevis testis by treatment with lysolecithin, which permeabilizes the sperm plasma membrane. 2. Egg extract is prepared by low speed centrifugation, addition of calcium to cause the extract to progress to interphase of the cell cycle, and a high-speed centrifugation to isolate interphase cytosol. 3. Nuclear transplantation: the nuclei and extract are combined with the linearized plasmid DNA to be introduced as the transgene and a small amount of restriction enzyme. During a short reaction, egg extract partially decondenses the sperm chromatin and the restriction enzyme generates chromosomal breaks that promote recombination of the transgene into the genome. The treated sperm nuclei are then transplanted into unfertilized eggs. Integration of the transgene usually occurs prior to the first embryonic cleavage such that the resulting embryos are not chimeric. These embryos can be analyzed without any need to breed to the next generation, allowing for efficient and rapid generation of transgenic embryos for analyses of promoter and gene function. Adult X. laevis resulting from this procedure also propagate the transgene through the germline and can be used to generate lines of transgenic animals for multiple purposes.

Production of Transgenic Xenopus laevis by Restriction Enzyme Mediated Integration and Nuclear Transplantation

This video protocol demonstrates a method for generating transgenic Xenopus laevis by introduction of transgenes into sperm nuclei followed by nuclear transplantation into unfertilized eggs.

Stable integration of cloned gene products into the Xenopus genome is necessary to control the time and place of expression, to express genes at later ...

Solid Plate-based Dietary Restriction in Caenorhabditis elegans

Reduction of food intake without malnutrition or starvation is known to increase lifespan and delay the onset of various age-related diseases in a wide range of species, including mammals. It also causes a decrease in body weight and fertility, as well as lower levels of plasma glucose, insulin, and IGF-1 in these animals. This treatment is often referred to as dietary restriction (DR) or caloric restriction (CR). The nematode Caenorhabditis elegans has emerged as an important model organism for studying the biology of aging. Both environmental and genetic manipulations have been used to model DR and have shown to extend lifespan in C. elegans. However, many of the reported DR studies in C. elegans were done by propagating animals in liquid media, while most of the genetic studies in the aging field were done on the standard solid agar in petri plates. Here we present a DR protocol using standard solid NGM agar-based plate with killed bacteria.

Solid Plate-based Dietary Restriction in Caenorhabditis elegans

Here we present a protocol for performing solid plate-based dietary restriction in C. elegans with killed bacteria.

Reduction of food intake without malnutrition or starvation is known to increase lifespan and delay the onset of various age-related diseases in a wide ...

High-throughput Yeast Plasmid Overexpression Screen

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, including those with direct relevance to human disease. Because of its short generation time and well-characterized genome, a major experimental advantage of the yeast model system is the ability to perform genetic screens to identify genes and pathways that are involved in a given process. Over the last thirty years such genetic screens have been used to elucidate the cell cycle, secretory pathway, and many more highly conserved aspects of eukaryotic cell biology 1-5. In the last few years, several genomewide libraries of yeast strains and plasmids have been generated 6-10. These collections now allow for the systematic interrogation of gene function using gain- and loss-of-function approaches 11-16. Here we provide a detailed protocol for the use of a high-throughput yeast transformation protocol with a liquid handling robot to perform a plasmid overexpression screen, using an arrayed library of 5,500 yeast plasmids. We have been using these screens to identify genetic modifiers of toxicity associated with the accumulation of aggregation-prone human neurodegenerative disease proteins. The methods presented here are readily adaptable to the study of other cellular phenotypes of interest.

Here we describe a plasmid overexpression screen in Saccharomyces cerevisiae, using an arrayed plasmid library and a high-throughput yeast transformation protocol with a liquid handling robot.

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, ...

Monitoring Plasmid Replication in Live Mammalian Cells over Multiple Generations by Fluorescence Microscopy

Few naturally-occurring plasmids are maintained in mammalian cells. Among these are genomes of gamma-herpesviruses, including Epstein-Barr virus (EBV) and Kaposi's Sarcoma-associated herpesvirus (KSHV), which cause multiple human malignancies 1-3. These two genomes are replicated in a licensed manner, each using a single viral protein and cellular replication machinery, and are passed to daughter cells during cell division despite their lacking traditional centromeres 4-8.
Much work has been done to characterize the replications of these plasmid genomes using methods such as Southern blotting and fluorescence in situ hybridization (FISH). These methods are limited, though. Quantitative PCR and Southern blots provide information about the average number of plasmids per cell in a population of cells. FISH is a single-cell assay that reveals both the average number and the distribution of plasmids per cell in the population of cells but is static, allowing no information about the parent or progeny of the examined cell.
Here, we describe a method for visualizing plasmids in live cells. This method is based on the binding of a fluorescently tagged lactose repressor protein to multiple sites in the plasmid of interest 9. The DNA of interest is engineered to include approximately 250 tandem repeats of the lactose operator (LacO) sequence. LacO is specifically bound by the lactose repressor protein (LacI), which can be fused to a fluorescent protein. The fusion protein can either be expressed from the engineered plasmid or introduced by a retroviral vector. In this way, the DNA molecules are fluorescently tagged and therefore become visible via fluorescence microscopy. The fusion protein is blocked from binding the plasmid DNA by culturing cells in the presence of IPTG until the plasmids are ready to be viewed.
This system allows the plasmids to be monitored in living cells through several generations, revealing properties of their synthesis and partitioning to daughter cells. Ideal cells are adherent, easily transfected, and have large nuclei. This technique has been used to determine that 84% of EBV-derived plasmids are synthesized each generation and 88% of the newly synthesized plasmids partition faithfully to daughter cells in HeLa cells. Pairs of these EBV plasmids were seen to be tethered to or associated with sister chromatids after their synthesis in S-phase until they were seen to separate as the sister chromatids separated in Anaphase10. The method is currently being used to study replication of KSHV genomes in HeLa cells and SLK cells. HeLa cells are immortalized human epithelial cells, and SLK cells are immortalized human endothelial cells. Though SLK cells were originally derived from a KSHV lesion, neither the HeLa nor SLK cell line naturally harbors KSHV genomes11. In addition to studying viral replication, this visualization technique can be used to investigate the effects of the addition, removal, or mutation of various DNA sequence elements on synthesis, localization, and partitioning of other recombinant plasmid DNAs.

A method of observing individual DNA molecules in live cells is described. The technique is based on the binding of a fluorescently tagged lac repressor protein to binding sites engineered into the DNA of interest. This method can be adapted to follow many recombinant DNAs in live cells over time.

Few naturally-occurring plasmids are maintained in mammalian cells.  Among these are genomes of gamma-herpesviruses, including Epstein-Barr virus  (EBV) ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Generation of Plasmid Vectors Expressing FLAG-tagged Proteins Under the Regulation of Human Elongation Factor-1&#945; Promoter Using Gibson Assembly

Gibson assembly (GA) cloning offers a rapid, reliable, and flexible alternative to conventional DNA cloning methods. We used GA to create customized plasmids for expression of exogenous genes in mouse embryonic stem cells (mESCs). Expression of exogenous genes under the control of the SV40 or human cytomegalovirus promoters diminishes quickly after transfection into mESCs. A remedy for this diminished expression is to use the human elongation factor-1 alpha (hEF1&#945;) promoter to drive gene expression. Plasmid vectors containing hEF1&#945; are not as widely available as SV40- or CMV-containing plasmids, especially those also containing N-terminal 3xFLAG-tags. The protocol described here is a rapid method to create plasmids expressing FLAG-tagged CstF-64 and CstF-64 mutant under the expressional regulation of the hEF1&#945; promoter. GA uses a blend of DNA exonuclease, DNA polymerase and DNA ligase to make cloning of overlapping ends of DNA fragments possible. Based on the template DNAs we had available, we designed our constructs to be assembled into a single sequence. Our design used four DNA fragments: pcDNA 3.1 vector backbone, hEF1&#945; promoter part 1, hEF1&#945; promoter part 2 (which contained 3xFLAG-tag purchased as a double-stranded synthetic DNA fragment), and either CstF-64 or specific CstF-64 mutant. The sequences of these fragments were uploaded to a primer generation tool to design appropriate PCR primers for generating the DNA fragments. After PCR, DNA fragments were mixed with the vector containing the selective marker and the GA cloning reaction was assembled. Plasmids from individual transformed bacterial colonies were isolated. Initial screen of the plasmids was done by restriction digestion, followed by sequencing. In conclusion, GA allowed us to create customized plasmids for gene expression in 5 days, including construct screens and verification.

Synthesis of custom plasmids is labor and time consuming. This protocol describes the use of Gibson assembly cloning to reduce the work and duration of custom DNA cloning procedure. The protocol described also produces reliable tagged protein constructs for mammalian expression at similar cost to the traditional cut-and-paste DNA cloning.

Gibson assembly (GA) cloning offers a rapid, reliable, and flexible alternative to conventional DNA cloning methods. We used GA to create customized ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...