Name: subcloning plus insertion (spi) - a novel recombineering method for the rapid construction of gene targeting vectors
Uploaded: 2015-01-08T13:00:00
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Description: Watch this Scientific Journal Video about Subcloning Plus Insertion SPI - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors at JoVE.com

T.R.R conceived the project, designed and performed experiments and prepared the manuscript. E.J.K and S.E.M performed experiments. All authors contributed intellectually to the final manuscript. This work was funded by a University of Leicester Innovation Fellowship to SEM and by a UK Medical Research Council (MRC) Senior Research Fellowship to S.M.C (MR/J009202/1). DNA sequencing was performed by Protein and Nucleic Acid Laboratories (PNACL), Centre for Core Biotechnology Services, University of Leicester. We wish to thank A. Francis Stewart for providing the Rox and Dre plasmids. pL451 and pL452 plasmids were obtained from National Cancer Institute (NCI), Frederick, USA.

1. Gene Targeting Design
<ol style="margin-left: 40px;">
	<li>Order an appropriate BAC clone covering the genomic region of interest. Ensure that this is isogenic to the type of ES cells to be modified e.g., RPCI-23 and RPCI-24 BAC clones for gene targeting with C57BL/6 ES cells.</li>
	<li>Apply conventional gene targeting criteria when designing the targeting vector. Key parameters include whether the modification is required to be constitutive or conditional, defining critical exons (CE) for deletion in a gene knockout strategy and spacing and placement of intronic cassettes. 
	NOTE: Each of these has been discussed in detail elsewhere10.</li>
	<li>Choose genomic regions each of 5-6 kb flanking the target modification site. 
	NOTE: The size of the subcloned insert is therefore typically 10-12 kb, although the upper limit can be as high as 80 kb with a low copy subcloning plasmid like p15A, pBR322 etc. and up to 200 kb with a BAC vector.</li>
</ol>
2. Multiplex Recombineering Oligos
<ol style="margin-left: 40px;">
	<li>Design oligos for the plasmid backbone (subcloning plasmid) and the selectable marker (insertion cassette). Design each oligo such that it contains 180 bp HA flanking the genomic target site and 20 bp of specific priming sequence of the insertion cassette or the subcloning plasmid (Figure 2). 
	NOTE: Homology arms should not contain any repetitive elements, presence of repetitive elements will result in incorrect targeting and subcloning. Repetitive elements can be detected by using web based tools such as the &#8216;&#8217;Repeat Masker&#8217;&#8217;.</li>
	<li>Include a unique restriction enzyme (RE) site on one of the vector oligos to linearize the gene targeting vector for ES cell targeting (Figure 2).</li>
	<li>Check oligo parameters using an oligo analyzer program. Take care to avoid secondary structures in the priming regions.</li>
	<li>Shorter HA (50 bp) of the insertion cassette is tolerated and yields lower number of recombinants but ensure that the subcloning plasmid HA is always longer (180 bp).</li>
	<li>Determine the orientation of the genomic DNA insert of the particular BAC clone using web based tools like &#8216;&#8217;cloneDB&#8217;&#8217;. Establish the direction of replication from OriS by checking the map of the BAC plasmid backbone used in the BAC library construction. 
	NOTE: OriS is usually opposite to the transcriptional direction of the Chloramphenicol (Chl) marker for all commonly used BAC plasmids.</li>
	<li>Add two terminal phosphorothioate (PTO) bonds to the 5&#8217; end of the oligo that is opposite to the direction of replication on the BAC clone. Add a 5&#8217; phosphate modification to the reverse oligo (Figure 2). 
	NOTE: The asymmetric phosphorothioated PCR cassette upon Red digestion generates an ssDNA intermediate that can prime the lagging strand of the replication fork. 
	NOTE: Maximal multiplex recombination frequency is observed with the lagging strand protected cassettes. Leading strand protected or dual protected cassettes may not produce equivalent recombination efficiencies at some loci.</li>
	<li>Order oligos with PAGE or HPLC purification.</li>
</ol>
3. BAC Clone Transformation with pSC101 BADgbaA Recombineering Plasmid.
<ol style="margin-left: 40px;">
	<li>To make the E. coli strain bearing the BAC recombineering proficient, transform it with the pSC101 BADgbaA plasmid containing the Red genes16. 
	NOTE: The pSC101 BADgbaA plasmid contains the Red and RecA genes under the control of the Arabinose inducible araC-PBAD promoter, a tetracycline selection marker and the temperature sensitive pSC101 replicon.
	<ol>
		<li>Stab a sterile pipette tip in the BAC agar culture and inoculate 5.0 ml of lysogeny broth (LB) pH 8.0 containing 12.5 &#956;g ml-1 Chl. Grow at 37 &#176;C for 5 hr&#160;shaking at 200 rpm.</li>
		<li>Chill 10% (v/v) glycerol solution, microcentrifuge tubes and electroporation cuvettes on ice. Cool a refrigerated large centrifuge and microcentrifuge to 4 &#176;C&#160;.</li>
		<li>Determine the optical density (OD) of the culture using a spectrophotometer and measure absorbance at 600 nm. Prepare electrocompetent cells (as described below) when an OD600 reading of 0.3 to 0.8 is reached.</li>
		<li>Spin down cells in a 50 ml centrifuge tube in a large centrifuge at 1,216 x&#160;g for 5 min at 4&#160;&#176;C&#160;.</li>
		<li>Wash cells with 1 ml of chilled 10% glycerol and spin down cells at 17,949 x&#160;g for 20 sec at 4 &#176;C&#160;. Perform the wash step a total of 3 times.</li>
		<li>Resuspend the cells in a total volume of 50 &#956;l of 10% glycerol and add 10-200 ng of the pSC101 BADgbaA recombineering plasmid. Obtain a single cell suspension by pipetting up and down several times and then transfer the cells to a pre-chilled 1 mm gap electroporation cuvette.</li>
		<li>Electroporate the cells with a setting of 1.8 kv, 25 &#956;F and 200 &#937;. 
		NOTE: Check the correct settings for each brand of electroporator. A time constant of electroporation less than 4 indicates the presence of salt and other impurities.</li>
		<li>Immediately recover the cells in 1 ml of LB and transfer the cells to a 50 ml centrifuge tube.</li>
		<li>Grow the BAC gbaA culture at 30&#160;&#176;C&#160;&#160;for 2 hr&#160;shaking at 200 rpm. 
		NOTE: The pSC101 BADgbaA plasmid is lost when cells are grown at 37&#160;&#176;C&#160;&#160;due to inactivation of the temperature sensitive RepE replication factor. Grow gbaA cells at 30&#160;&#176;C&#160;&#160;to maintain the recombineering functions.</li>
		<li>Add 9 ml of LB containing 12.5 &#956;g ml-1 Chl and 4 &#956;g ml-1 Tetracycline (Tet) to the recovered BAC gbaA culture. Grow the BAC gbaA culture O/N&#160;at 30&#160;&#176;C&#160;&#160;shaking at 200 rpm. 
		NOTE: The transformation efficiency of electroporating the supercoiled gbaA plasmid is sufficiently high to permit saturation growth O/N&#160;in liquid media.</li>
	</ol>
	</li>
</ol>
4. Preparation of Insertion Cassettes and Subcloning Plasmids
<ol style="margin-left: 40px;">
	<li>To incorporate HA into the insertion cassette(s) and the subcloning plasmid, perform polymerase chain reaction (PCR) using the long modified oligos as described below.
	<ol>
		<li>Optional: to prevent plasmid carryover into the recombineering reaction, use an R6K origin or similar narrow host range plasmid template to amplify the insertion cassette.</li>
		<li>Alternatively, linearize the plasmid template using an RE digest (Table 2). Choose a RE that cuts outside the PCR amplification region and is heat inactivated. Heat inactivate the RE as recommended by the manufacturer.</li>
		<li>Set up polymerase chain reaction (PCR) using a high-fidelity hotstart DNA polymerase system. Prepare a PCR master mix as detailed (Table 3). Perform thermal cycling as shown (Table 3).</li>
	</ol>
	</li>
	<li>Analyze PCR products by agarose gel electrophoresis. Load 1-5 &#956;l&#160;of each PCR onto a 1% (w/v) agarose gel containing 0.5 mg ml-1 Ethidium Bromide (EtBR). 
	NOTE: The presence of non-specific amplification products does not interfere in the recombineering reaction. In some cases however, primer-dimers may decrease recombineering efficiency.</li>
	<li>Purify PCR products using a PCR purification kit.
	<ol>
		<li>Optional: remove the plasmid template from PCRs by treatment with DpnI followed by PCR clean-up. Elute DNA in a minimal volume of sterile deionized water (as recommended by the manufacturer). 
		NOTE: Addition of DpnI to unpurified PCR reactions results in reduced efficiency of cleavage of the methylated plasmid DNA template.</li>
	</ol>
	</li>
	<li>Quantify PCR amplified DNA by agarose gel analysis against a known set of DNA standards e.g., &#955;&#160;HindIII digest or by using a nanodrop spectrophotometer.</li>
</ol>
5. Subcloning Plus Insertion
<ol style="margin-left: 40px;">
	<li>Dilute the O/N BAC gbaA culture 50 fold by adding 200&#160;&#956;l&#160;in 10 ml LB+Chl+Tet. Grow at 30&#160;&#176;C&#160;&#160;shaking at 200 rpm&#160;for 1 hr 50 min. Include an sample to be used as negative control.</li>
	<li>Chill all recombineering materials and equipment to 4&#160;&#176;C&#160;&#160;as described in step 3.1.2.</li>
	<li>Pour LB agar pH 8 plates containing the correct concentration of the appropriate selective antibiotic(s) that will allow selection of the DNA fragment that is inserted as well as for the selection of the subcloning vector (Table 4). 
	NOTE: Some antibiotics are pH sensitive. Use LB agar pH 8 as a rule.</li>
	<li>Prepare a 10% (w/v) solution of L-Arabinose. Filter sterilize through a 0.2 &#956;m syringe filter. 
	NOTE: Arabinose induces the expression of the recombineering proteins from the gbaA plasmid.</li>
	<li>Check the OD of the BAC gbaA culture using a spectrophotometer. Once an OD600 of 0.25-0.3 is reached, induce the recombineering proteins as described in the following step.</li>
	<li>Add 200 &#956;l&#160;of the 10% Arabinose solution to 10 ml of the BAC culture to achieve a final concentration of Arabinose of 0.2%. Include an uninduced culture (without Arabinose) to be used as negative control.</li>
	<li>Transfer the BAC culture to a 37&#160;&#176;C&#160;&#160;shaking incubator and induce Red expression for 45 min shaking at 230 rpm. 
	NOTE: Expression of Red proteins is inefficient at 30&#160;&#176;C&#160;.</li>
	<li>Spin down cells and wash with 10% glycerol 3 times as described in step 3.1.5.</li>
	<li>Add 600-1,000 ng each of the subcloning plasmid and the insertion cassette(s) to the recombineering reaction. Include a vector only and vector plus single insert controls to check the recombination proficiency and integrity of the vector and the cassettes.</li>
	<li>Obtain a single cell suspension by pipetting up and down. Perform electroporation as described in step 3.1.7 and subsequent recovery in 1 ml LB at 37&#160;&#176;C&#160;&#160;for 1 hr for multi-copy plasmids or in 10 ml of LB at 37&#160;&#176;C&#160;&#160;for 3 hr&#160;for BAC vectors.</li>
	<li>Plate different dilutions of the recovered culture e.g., 90%, 10%, 1% on the dual selection agar plates and grow at 37&#160;&#176;C&#160;&#160;for 16 hr.</li>
</ol>
6. Analysis of Recombinants
<ol style="margin-left: 40px;">
	<li>Optional: Pick 6 to 12 colonies and perform colony PCR using a HA flanking primer and an insert specific primer. Include the subcloning plasmid and the insertion cassette plasmid as well as the parent BAC clone as negative controls.
	<ol>
		<li>Perform agarose gel analysis of the colony PCRs. Identify positive clones by the presence of a bright band at the expected size. 
		NOTE: The high efficiency of the SPI cloning process generates mostly correct recombinants.</li>
	</ol>
	</li>
	<li>Pick colonies into 5 ml LB pH 8 containing the selective antibiotic and grow O/N&#160;at 37&#160;&#176;C&#160;.</li>
	<li>Prepare DNA minipreps using a column purification kit as per the manufacturer&#8217;s instructions.</li>
	<li>Prepare BAC miniprep DNA using standard phenol-chloroform isolation or a similar protocol.</li>
	<li>Perform RE digests on the miniprep DNA. Separate DNA by agarose gel electrophoresis. Analyze RE patterns to identify the clones containing the expected fragments sizes of the correct targeting vector. Choose an RE that clearly discriminates between the vector lacking insert(s) and the vector containing the insert(s).</li>
	<li>Optional: to obtain cells that are devoid of the BAC, which is still present in the E. coli after recombineering, use the miniprep plasmid DNA to transform DH5alpha or DH10B E. coli cells.</li>
	<li>Perform DNA sequencing across the HA and insertion cassette to check oligo synthesis errors.</li>
</ol>

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L., Jenkins, N. A., Copeland, N. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple+and+highly+efficient+BAC+recombineering+using+galK+selection.">Simple and highly efficient BAC recombineering using galK selection.</a> Nucleic Acids Res. 33, e36 (2005).</li><li>Cong, L., 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=Multiplex+Genome+Engineering+Using+CRISPR/Cas+Systems.">Multiplex Genome Engineering Using CRISPR/Cas Systems.</a> Science. 339, 819-823 (2013).</li></ol>

T.R.R is the inventor of a British patent application on multiplex recombineering technology.

Construction of ES cell lines and mouse models has historically involved gene targeting using plasmid constructs that contained the modified allele4. However, the construction of these complex gene targeting vectors has proved to be a significant bottleneck in the timely production of such models. The development of recombineering based vector construction strategies has allowed improved vector designs and more efficient vector assembly. Nonetheless, current recombineering protocols still involve multiple steps, require intermediate plasmid purification and use different bacterial strains. Subcloning plus insertion offers a novel approach to vector construction that can be performed in one electroporation event in the resident BAC host strain. The utility of SPI in gene targeting was tested here in a variety of vector construction applications. In all instances examined here, SPI proved to be efficient and the correct recombinant plasmid was produced. In the majority of the cases, the multiple different cassettes were correctly inserted in the targeting vector. Large DNA cassettes and vectors were easily accommodated in the SPI protocol and demonstrated the flexibility of this system.
SPI relies on the use of long homology sequences and phosphorothioate (PTO) protection of the linear DNA cassettes. PTO modification confers protection against exonucleases to the linear DNA20,21 and the long HA increases recombination efficiency to permit multiplexing. However, synthesis of longer oligo sequences increases the chances of accumulating errors especially deletions. Mutations in the oligo can be particularly detrimental if they cover protein coding regions. DNA sequencing across the HA and covering the full-length of the inserted cassette is highly recommended to eliminate clones with any sequence alterations. Use of a high-fidelity DNA polymerase system is also suggested to avoid introduction of any PCR errors. The length and composition of the HA of the subcloning plasmid is more critical relative to that of the insertion cassette (data not shown). For particularly sensitive applications like construction of knockin vectors, where any mutations in exon regions are not tolerated, the HA of the insertion cassette can be shortened (50-120 bp) to avoid problems associated with long oligos. The insertion cassette can also be left unmodified or dual phosphorothioated (where knowledge of the direction of replication is not available). But multiplexing in these cases still requires long protected HA subcloning plasmids. A caveat of this particular strategy is the lowering of multiplexing efficiency that can potentially impact SPI cloning at different loci.
The length of the insertion cassette and the subcloning plasmid is another important parameter in SPI cloning. Larger DNA molecules electroporate less efficiently and the effect is cumulative, given the requirement to introduce all the cassettes in the same cell (data not shown). Multiplexing is most efficient with smaller insertion cassettes. DNA fragments larger than 3 kb also place a limit on Red mediated ssDNA processing, which is most efficient up to 3 kb30. Insertion cassettes exceeding 3 kb are dual resected and recombine less efficiently via a beta independent pathway20. Therefore, screening of sufficient colonies to identify the correct clone becomes important in these cases. A longer duration of recombination post electroporation also increases the chances of recovery of the correct clone in difficult SPI exercises. Up to four small cassettes (&#60; 1.5 kb) can be inserted simultaneously with the SPI process, though the multiplexing efficiency decreases with each additional cassette (data not shown). However, this reflects the outcome of an optimal SPI experiment and it is advisable to consider the limits of multiplexing when using many large cassettes.
The development of genome editing tools like clustered regularly interspaced short palindromic repeats (CRISPR)-cas9 endonuclease system has enabled the creation of novel genome modifications and has led to more efficient genome engineering36. However, these newer technologies have complemented gene targeting vectors rather than supplanted them. It is envisaged that the CRISPR-cas system could replace antibiotic selection and further refine the multiplex recombineering protocol.

The development of gene targeting technologies has enabled the construction of cell lines and mouse models to investigate different biological systems1-3. A key first stage in the modification of a genomic sequence is the design and construction of a gene targeting vector4. Targeting vectors are plasmid constructs that carry the allele of interest containing the desired modifications(s) flanked by a selection marker (e.g.,&#160;neomycin), and long genomic regions required for efficient homologous recombination in mammalian cells5. Precise gene modification is achieved by the introduction of the targeting vector into embryonic stem (ES) cells6 or somatic cells7, whereby homologous recombination between identical stretches of DNA sequence on the targeting vector and the genomic locus results in the transfer of the intended modification to the genome by gene conversion8. Such modified ES cells can be injected into mouse blastocysts to produce offspring (chimaeras) that can then transmit these modified alleles via the germline9. An alternate route to produce transgenic mice involves the microinjection of a gene expression vector into single celled mouse zygotes, which leads to the random integration of the vector in the mouse genome10. Traditional methods of vector construction have relied on conventional &#8216;cut and paste&#8217; cloning using restriction enzymes and DNA ligases to clone the different selection marker and genomic fragments into a vector backbone. However, an inherent limiting factor of traditional cloning is the positioning and choice of restriction sites, especially with longer DNA sequences. This often necessitates multiple subcloning steps and also introduces extraneous DNA sequences into the vector, which often leads to a lower efficiency of gene targeting.
Recombineering (recombinogenic engineering) is a DNA engineering technology that overcomes these limitations by using homologous recombination (HR) mediated by phage recombination proteins in E. coli cells11,12. Since any region of a homologous sequence can serve as a substrate of recombineering, the constraints of availability of restriction sites are removed. Large DNA sequences can be seamlessly modified directly in vivo, thus also preserving their structural integrity13. Recombineering is very efficient with short homologies (50 bp)14 and therefore homology arms (HA) can be conveniently incorporated into synthetic oligo sequences. In a typical recombineering experiment, an oligo or a double stranded DNA (dsDNA) fragment containing HA is electroporated into recombineering competent E. coli cells containing the target located either on the chromosome or on a plasmid15. The recombination potential is conferred by inducible expression of the Red recombination proteins of the phage16,17 or the RecET proteins of the rac prophage18. The Red/RecE exonuclease converts linear dsDNA to a single-stranded DNA (ssDNA) intermediate, which is then bound by its partner, Red/RecT, a single-stranded annealing protein (SSAP)19. The annealing of a long ssDNA or a short oligo to its complementary target sequence occurs on the lagging strand of the replication fork and leads to the incorporation of the sequence at the target site. Lagging strand ssDNA recombination is the basis of the high efficiency of recombineering and can be described by the &#8216;beta&#8217; recombination model20,21.
A typical recombineering workflow to build a gene targeting vector involves either of the two following routes. One route involves subcloning the desired genomic region from a mouse BAC clone into a plasmid followed by the sequential insertion of LoxP recombination sites, a selection marker22 etc., or the alternative route involves targeting the BAC genomic locus with the different targeting vector elements by multiple rounds of recombineering10,23 and then subcloning the modified locus into a plasmid by gap repair cloning24,25. Variations on this theme have been used in different high-throughput recombineering pipelines as part of large mouse production programs26,27. However, these procedures involve complicated and lengthy stages, require the use of specialized vectors and E. coli strains (e.g., Cre expressing cells) and utilize one or more intermediate steps of vector DNA purification and re-transformation (Table 1). Subcloning plus insertion (SPI) is a novel recombineering technique that combines beta recombination and gap repair cloning into a single process (Figure 1). SPI vector assembly is simple, quick and flexible and offers significant improvement on standard recombineering approaches (Table 1). Here, we demonstrate the ease and utility of using SPI for different vector construction applications with a particular emphasis on constructing non-standard and challenging vector designs. Test cases included the construction of a fluorescent reporter knockin vector, a dual tagged protein expression knockin vector, a BAC fluorescent reporter vector and a conditional knockout vector. These examined variously the requirement to localize a cell surface receptor, purify a nuclear protein complex or conditionally ablate the expression of a gene.

<table border="0" cellpadding="0" cellspacing="0" width="1597">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:315px;">Name of Material/ Equipment</td>
			<td style="width:127px;">Company</td>
			<td style="width:216px;">Catalog Number</td>
			<td style="width:941px;">Comments/Description</td>
		</tr>
		<tr height="210">
			<td height="210" style="height:210px;width:315px;">Antibiotics except Zeocin</td>
			<td style="width:127px;">Sigma: http://www.sigmaaldrich.com</td>
			<td style="width:216px;">Ampicillin, A9518-5G; 
			Chloramphenicol , C1919-5G; Blasticidin, 15205-25MG; Gentamicin, G1264-50MG; Hygromycin, H3274-50MG; Kanamycin,: K1876-1G; 
			Tetracycline hydrochloride, T7660-5G; Trimethoprim, 92131-1G</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:315px;">Zeocin</td>
			<td style="width:127px;">Invivogen:&#160;</td>
			<td style="width:216px;">ant-zn-1</td>
			<td>Zeocin is also available from Life technologies/Fisher</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:315px;">C57BL/6 BAC clones</td>
			<td style="width:127px;">CHORI: https://bacpac.chori.org/&#160;</td>
			<td style="width:216px;">RPCI-23 or RPCI-24 BAC libraries</td>
			<td>129/Sv BAC clones can be obtained from Invivogen or Life technologies/Fisher</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:315px;">KOD hotstart DNA polymerase system</td>
			<td style="width:127px;">Millipore: https://www.merckmillipore.com</td>
			<td style="width:216px;">71086-3</td>
			<td></td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:315px;">L-Arabinose</td>
			<td style="width:127px;">Sigma: http://www.sigmaaldrich.com</td>
			<td style="width:216px;">A3256-25G</td>
			<td></td>
		</tr>
		<tr height="189">
			<td height="189" style="height:189px;width:315px;">Long oligos</td>
			<td style="width:127px;">IDT: https://www.idtdna.com; Gene link: https://www.genelink.com</td>
			<td style="width:216px;">IDT Ultramers or Gene Link long oligos</td>
			<td>PTO and Phosphosphate available as custom modifications. PAGE purification strongly recommended for long oligos.</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:315px;">MinElute PCR purification kit</td>
			<td style="width:127px;">Qiagen: http://www.qiagen.com&#160;</td>
			<td align="right" style="width:216px;">28004</td>
			<td>Minimum elution PCR purifications kits are preferred.</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:315px;">QIAPrep Spin Mini Prep kit</td>
			<td style="width:127px;">Qiagen: http://www.qiagen.com&#160;</td>
			<td align="right" style="width:216px;">27104</td>
			<td>Silica column or similar matrix kits are preferred.</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:315px;">Restriction enzymes</td>
			<td style="width:127px;">NEB: https://www.neb.com</td>
			<td style="width:216px;">DpnI: R0176L</td>
			<td>See NEB website for a list of RE.</td>
		</tr>
	</tbody>
</table>

subcloning plus insertion (spi) - a novel recombineering method for the rapid construction of gene targeting vectors

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

Watch this Scientific Journal Video about Subcloning Plus Insertion SPI - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors at JoVE.com

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

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

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

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

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Research

JoVE Journal

Biology

Hi-C: A Method to Study the Three-dimensional Architecture of Genomes.

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, into close spatial proximity 2-6. Deciphering the relationship between chromosome organization and genome activity will aid in understanding genomic processes, like transcription and replication. However, little is known about how chromosomes fold. Microscopy is unable to distinguish large numbers of loci simultaneously or at high resolution. To date, the detection of chromosomal interactions using chromosome conformation capture (3C) and its subsequent adaptations required the choice of a set of target loci, making genome-wide studies impossible 7-10.
We developed Hi-C, an extension of 3C that is capable of identifying long range interactions in an unbiased, genome-wide fashion. In Hi-C, cells are fixed with formaldehyde, causing interacting loci to be bound to one another by means of covalent DNA-protein cross-links. When the DNA is subsequently fragmented with a restriction enzyme, these loci remain linked. A biotinylated residue is incorporated as the 5' overhangs are filled in. Next, blunt-end ligation is performed under dilute conditions that favor ligation events between cross-linked DNA fragments. This results in a genome-wide library of ligation products, corresponding to pairs of fragments that were originally in close proximity to each other in the nucleus. Each ligation product is marked with biotin at the site of the junction. The library is sheared, and the junctions are pulled-down with streptavidin beads. The purified junctions can subsequently be analyzed using a high-throughput sequencer, resulting in a catalog of interacting fragments.
Direct analysis of the resulting contact matrix reveals numerous features of genomic organization, such as the presence of chromosome territories and the preferential association of small gene-rich chromosomes. Correlation analysis can be applied to the contact matrix, demonstrating that the human genome is segregated into two compartments: a less densely packed compartment containing open, accessible, and active chromatin and a more dense compartment containing closed, inaccessible, and inactive chromatin regions. Finally, ensemble analysis of the contact matrix, coupled with theoretical derivations and computational simulations, revealed that at the megabase scale Hi-C reveals features consistent with a fractal globule conformation.

The Hi-C method allows unbiased, genome-wide identification of chromatin interactions (1). Hi-C couples proximity ligation and massively parallel sequencing. The resulting data can be used to study genomic architecture at multiple scales: initial results identified features such as chromosome territories, segregation of open and closed chromatin, and chromatin structure at the megabase scale.

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, ...

The Production of C. elegans Transgenes via Recombineering with the galK Selectable Marker

The creation of transgenic animals is widely utilized in C. elegans research including the use of GFP fusion proteins to study the regulation and expression pattern of genes of interest or generation of tandem affinity purification (TAP) tagged versions of specific genes to facilitate their purification. Typically transgenes are generated by placing a promoter upstream of a GFP reporter gene or cDNA of interest, and this often produces a representative expression pattern. However, critical elements of gene regulation, such as control elements in the 3' untranslated region or alternative promoters, could be missed by this approach. Further only a single splice variant can be usually studied by this means. In contrast, the use of worm genomic DNA carried by fosmid DNA clones likely includes most if not all elements involved in gene regulation in vivo which permits the greater ability to capture the genuine expression pattern and timing. To facilitate the generation of transgenes using fosmid DNA, we describe an E. coli based recombineering procedure to insert GFP, a TAP-tag, or other sequences of interest into any location in the gene. The procedure uses the galK gene as the selection marker for both the positive and negative selection steps in recombineering which results in obtaining the desired modification with high efficiency. Further, plasmids containing the galK gene flanked by homology arms to commonly used GFP and TAP fusion genes are available which reduce the cost of oligos by 50% when generating a GFP or TAP fusion protein. These plasmids use the R6K replication origin which precludes the need for extensive PCR product purification. Finally, we also demonstrate a technique to integrate the unc-119 marker on to the fosmid backbone which allows the fosmid to be directly injected or bombarded into worms to generate transgenic animals. This video demonstrates the procedures involved in generating a transgene via recombineering using this method.

The Production of C. elegans Transgenes via Recombineering with the galK Selectable Marker

The ability to produce transgenes for Caenorhabditis elegans using genomic DNA carried by fosmids is particularly attractive as all of the native regulatory elements are retained. Described is a simple and robust procedure for the production of transgenes via recombineering with the galK selectable marker.

The creation of transgenic animals is widely utilized in C. elegans research including the use of GFP fusion proteins to study the regulation and ...

A Novel Method for the Culture and Polarized Stimulation of Human Intestinal Mucosa Explants

Few models currently exist to realistically simulate the complex human intestine's micro-environment, where a variety of interactions take place. Proper homeostasis directly depends on these interactions, as they shape an entire immunological response inducing tolerance against food antigens while at the same time mounting effective immune responses against pathogenic microbes accidentally ingested with food.
Intestinal homeostasis is preserved also through various complex interactions between the microbiota (including food-associated beneficial bacterial strains) and the host, that regulate the attachment/degradation of mucus, the production of antimicrobial peptides by the epithelial barrier, and the "education" of epithelial cells' that controls the tolerogenic or immunogenic phenotype of unique, gut-resident lymphoid cells' populations. These interactions have been so far very difficult to reproduce with in vitro assays using either cultured cell lines or peripheral blood mononuclear cells. In addition, mouse models differ substantially in components of the intestinal mucosa (mucus layer organization, commensal bacteria community) with respect to the human gut. Thus, studies of a variety of treatments to be brought in the clinics for important stress-related or pathological conditions such as irritable bowel syndrome, inflammatory bowel disease or colorectal cancer have been difficult to carry out.
To address these issues, we developed a novel system that enables us to stimulate explants of human intestinal mucosa that retain their in situ conditioning by the host microbiota and immune response, in a polarized fashion. Polarized apical stimulation is of great importance for the outcome of the elicited immune response. It has been repeatedly shown that the same stimuli can produce completely different responses when they bypass the apical face of the intestinal epithelium, stimulating epithelial cells basolaterally or coming into direct contact with lamina propria components, switching the phenotype from tolerogenic to immunogenic and causing unnecessary and excessive inflammation in the area.
We achieved polarized stimulation by gluing a cave cylinder which delimited the area of stimulation on the apical face of the mucosa as will be described in the protocol. We used this model to examine, among others, differential effects of three different Lactobacilli strains. We show that this model system is very powerful to assess the immunomodulatory properties of probiotics in healthy and disease conditions.

We introduce a novel method for the maintenance of human intestinal mucosa in culture and monitoring of the response to various types of stimuli over at least 24 hrs. With our method, the polarity of the tissue is maintained, allowing for a physiological stimulation via the apical route.

Few models currently exist to realistically simulate the complex human intestine's micro-environment, where a variety of interactions take place. Proper ...

Recombineering Homologous Recombination Constructs in Drosophila

The continued development of techniques for fast, large-scale manipulation of endogenous gene loci will broaden the use of Drosophila melanogaster as a genetic model organism for human-disease related research. Recent years have seen technical advancements like homologous recombination and recombineering. However, generating unequivocal null mutations or tagging endogenous proteins remains a substantial effort for most genes. Here, we describe and demonstrate techniques for using recombineering-based cloning methods to generate vectors that can be used to target and manipulate endogenous loci in vivo. Specifically, we have established a combination of three technologies: (1) BAC transgenesis/recombineering, (2) ends-out homologous recombination and (3) Gateway technology to provide a robust, efficient and flexible method for manipulating endogenous genomic loci. In this protocol, we provide step-by-step details about how to (1) design individual vectors, (2) how to clone large fragments of genomic DNA into the homologous recombination vector using gap repair, and (3) how to replace or tag genes of interest within these vectors using a second round of recombineering. Finally, we will also provide a protocol for how to mobilize these cassettes in vivo to generate a knockout, or a tagged gene via knock-in. These methods can easily be adopted for multiple targets in parallel and provide a means for manipulating the Drosophila genome in a timely and efficient manner.

Recombineering Homologous Recombination Constructs in Drosophila

Homologous recombination techniques greatly advance Drosophila genetics by enabling the creation of molecularly precise mutations. The recent adoption of recombineering allows one to manipulate large pieces of DNA and transform them into Drosophila6. The methods presented here combine these techniques to rapidly generate large homologous recombination vectors.

The continued development of techniques for fast, large-scale manipulation of endogenous gene loci will broaden the use of Drosophila melanogaster as a ...

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. Thus, protocols to isolate and culture individual organs of interest are essential to provide a method of both visualizing changes in development and allowing novel treatment strategies. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel. This substrate provides enough support to maintain the three-dimensional structure but is flexible enough to allow continued contraction. In brief, hearts are excised from the embryo and placed in a mixture of cold Matrigel diluted 1:1 with growth medium. After the diluted Matrigel solidifies, growth medium is added to the culture dish. Hearts excised as late as embryonic day 16.5 were viable for four days post-dissection. Analysis of the coronary plexus shows that this method does not disrupt coronary vascular development. Thus, we present a novel method for long-term culture of embryonic hearts.

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel.

Developmental studies in the mouse are hampered  by the inaccessibility of the embryo during gestation. Thus, protocols to  isolate and culture individual ...

Zinc-finger Nuclease Enhanced Gene Targeting in Human Embryonic Stem Cells

One major limitation with current human embryonic stem cell (ESC) differentiation protocols is the generation of heterogeneous cell populations.&#160;These cultures contain the cells of interest, but are also contaminated with undifferentiated ESCs, non-neural derivatives and other neuronal subtypes.&#160; This limits their use in in vitro and in vivo applications, such as in vitro modeling for drug discovery or cell replacement therapy. To help overcome this, reporter cell lines, which offer a means to visualize, track and isolate cells of interest, can be engineered.&#160;However, to achieve this in human embryonic stem cells via conventional homologous recombination is extremely inefficient.&#160;This protocol describes targeting of the Pituitary homeobox 3 (PITX3) locus in human embryonic stem cells using custom designed zinc-finger nucleases, which introduce site-specific double-strand DNA breaks, together with a PITX3-EGFP-specific DNA donor vector. Following the generation of the PITX3 reporter cell line, it can then be differentiated using published protocols for use in studies such as in vitro Parkinson&#8217;s disease modeling or cell replacement therapy.

Reporter cell lines offer a means to visualize, track and isolate cells of interest from heterogeneous populations.&#160;However, gene-targeting using conventional homologous recombination in human embryonic stem cells is extremely inefficient. Herein, we describe targeting CNS midbrain specific transcription factor PITX3 locus with EGFP using zinc-finger nuclease enhanced homologous recombination.

One major limitation with current human embryonic stem cell (ESC) differentiation protocols is the generation of heterogeneous cell ...

Filtration Isolation of Nucleic Acids:  A Simple and Rapid DNA Extraction Method

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad to extract cellular DNA from whole blood in less than 2 min. The blood specimen is treated with detergent, mixed briefly and applied by pipet to the separation membrane. The lysate wicks into the blotting pad due to capillary action, capturing the genomic DNA on the surface of the separation membrane. The extracted DNA is retained on the membrane during a simple wash step wherein PCR inhibitors are wicked into the absorbent blotting pad. The membrane containing the entrapped DNA is then added to the PCR reaction without further purification. This simple method does not require laboratory equipment and can be easily implemented with inexpensive laboratory supplies. Here we describe a protocol for highly sensitive detection and quantitation of HIV-1 proviral DNA from 100 &#181;l whole blood as a model for early infant diagnosis of HIV that could readily be adapted to other genetic targets.

We describe here a simple and rapid paper-based DNA extraction method of HIV proviral DNA from whole blood detected by quantitative PCR. This protocol can be extended for use in detecting other genetic markers or using alternative amplification methods.

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad ...

A Rapid, Scalable Method for the Isolation, Functional Study, and Analysis of Cell-derived Extracellular Matrix

The extracellular matrix (ECM) is recognized as a diverse, dynamic, and complex environment that is involved in multiple cell-physiological and pathological processes. However, the isolation of ECM, from tissues or cell culture, is complicated by the insoluble and cross-linked nature of the assembled ECM and by the potential contamination of ECM extracts with cell surface and intracellular proteins. Here, we describe a method for use with cultured cells that is rapid and reliably removes cells to isolate a cell-derived ECM for downstream experimentation.
Through use of this method, the isolated ECM and its components can be visualized by in situ immunofluorescence microscopy. The dynamics of specific ECM proteins can be tracked by tracing the deposition of a tagged protein using fluorescence microscopy, both before and after the removal of cells. Alternatively, the isolated ECM can be extracted for biochemical analysis, such as sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. At larger scales, a full proteomics analysis of the isolated ECM by mass spectrometry can be conducted. By conducting ECM isolation under sterile conditions, sterile ECM layers can be obtained for functional or phenotypic studies with any cell of interest. The method can be applied to any adherent cell type, is relatively easy to perform, and can be linked to a wide repertoire of experimental designs.

The extracellular matrix plays a major role in defining the microenvironment of cells and in modulating cell behavior and phenotype. We describe a rapid method for the isolation of cell-derived extracellular matrix, which can be adapted to different scales for microscopic, biochemical, proteomic, or functional studies.

The extracellular matrix (ECM) is recognized as a diverse, dynamic, and complex environment that is involved in multiple cell-physiological and ...

A Simple and Efficient Method for In Vivo Cardiac-specific Gene Manipulation by Intramyocardial Injection in Mice

Gene manipulation specifically in the heart significantly potentiate the investigation of cardiac disease pathomechanisms and their therapeutic potential. In vivo cardiac-specific gene delivery is commonly achieved by either systemic or local delivery. Systemic injection via tail vein is easy and efficient in manipulating cardiac gene expression by using recombinant adeno-associated virus 9 (AAV9). However, this method requires a relatively high amount of vector for efficient transduction, and may result in nontarget organ gene transduction. Here, we describe a simple, efficient, and time-saving method of intramyocardial injection for in vivo cardiac-specific gene manipulation in mice. Under anesthesia (without ventilation), the pectoral major and minor muscles were bluntly dissected, and the mouse heart was quickly exposed by manual externalization through a small incision at the fourth intercostal space. Subsequently, adenovirus encoding luciferase (Luc) and vitamin D receptor (VDR), or short hairpin RNA (shRNA) targeting VDR, was injected with a Hamilton syringe into the myocardium. Subsequent in vivo imaging demonstrated that luciferase was successfully overexpressed specifically in the heart. Moreover, Western blot analysis confirmed the successful overexpression or silencing of VDR in the mouse heart. Once mastered, this technique can be used for gene manipulation, as well as injection of cells or other materials such as nanogels in the mouse heart.

A Simple and Efficient Method for In Vivo Cardiac-specific Gene Manipulation by Intramyocardial Injection in Mice

Here we present a protocol for cardiac-specific gene manipulation in mice. Under anesthesia, the mouse hearts were externalized through the fourth intercostal space. Subsequently, adenoviruses encoding specific genes were injected with a syringe into the myocardium, followed by protein expression measurement via in vivo imaging and Western blot analysis.

Gene manipulation specifically in the heart significantly potentiate the investigation of cardiac disease pathomechanisms and their therapeutic potential. ...

Protocols for Testing the Toxicity of Novel Insecticidal Chemistries to Mosquitoes

New classes of insecticides with novel modes of action are needed to control insecticide resistant populations of mosquitoes that transmit diseases such as Zika, dengue and malaria. Assays for rapid, high-throughput analyses of unformulated novel chemistries against mosquito larvae and adults are presented. We describe protocols for single point-dose and dose response assays to evaluate the toxicity of small molecule chemistries to the Aedes aegypti vector of Zika, dengue and yellow fever,&#160;the malaria vector, Anopheles gambiae and the northern house mosquito, Culex quinquefasciatus, on contact and via ingestion. As an example, we evaluated the toxicity of amitriptyline, a small molecule antagonist of G protein-coupled receptors, via larval, adult topical and adult blood-feeding assay. The protocols provide a starting point to investigate insecticide potential. Results are discussed in the context of additional experiments to explore product applications and mechanisms for delivery.

Protocols are described for assessing the toxicity of chemistries to immature and adult mosquitoes for development as new classes of larvicides, adulticides and endectocides. The protocols enable high-throughput testing of multiple chemistries at single-point dose and subsequent evaluation via dose response assay to determine toxicity on contact or via ingestion.

New classes of insecticides with novel modes of action are needed to control insecticide resistant populations of mosquitoes that transmit diseases such ...