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><tbody><tr><td>Antibiotics except Zeocin</td><td>Sigma: http://www.sigmaaldrich.com</td><td>Ampicillin, A9518-5G;&lt;br/&gt; Chloramphenicol , C1919-5G; Blasticidin, 15205-25MG; Gentamicin, G1264-50MG; Hygromycin, H3274-50MG; Kanamycin,: K1876-1G;&lt;br/&gt; Tetracycline hydrochloride, T7660-5G; Trimethoprim, 92131-1G</td><td></td></tr><tr><td>Zeocin</td><td>Invivogen:&amp;nbsp;</td><td>ant-zn-1</td><td>Zeocin is also available from Life technologies/Fisher</td></tr><tr><td>C57BL/6 BAC clones</td><td>CHORI: https://bacpac.chori.org/&amp;nbsp;</td><td>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><td>KOD hotstart DNA polymerase system</td><td>Millipore: https://www.merckmillipore.com</td><td>71086-3</td><td></td></tr><tr><td>L-Arabinose</td><td>Sigma: http://www.sigmaaldrich.com</td><td>A3256-25G</td><td></td></tr><tr><td>Long oligos</td><td>IDT: https://www.idtdna.com; Gene link: https://www.genelink.com</td><td>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><td>MinElute PCR purification kit</td><td>Qiagen: http://www.qiagen.com&amp;nbsp;</td><td>28004</td><td>Minimum elution PCR purifications kits are preferred.</td></tr><tr><td>QIAPrep Spin Mini Prep kit</td><td>Qiagen: http://www.qiagen.com&amp;nbsp;</td><td>27104</td><td>Silica column or similar matrix kits are preferred.</td></tr><tr><td>Restriction enzymes</td><td>NEB: https://www.neb.com</td><td>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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Biology

16.5K Views.  University of Leicester. 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 ‘subcloning plus insertion’, which can be used to generate custom gene targeting vectors rapidly.

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

Transfection, Selection, and Colony-picking of Human Induced Pluripotent Stem Cells TALEN-targeted with a GFP Gene into the AAVS1 Safe Harbor

Targeted transgene addition can provide persistent gene expression while circumventing the gene silencing and insertional mutagenesis caused by viral vector mediated random integration. This protocol describes a universal and efficient transgene targeted addition platform in human iPSCs based on utilization of validated open-source TALENs and a gene-trap-like donor to deliver transgenes into a safe harbor locus. Importantly, effective gene editing is rate-limited by the delivery efficiency of gene editing vectors. Therefore, this protocol first focuses on preparation of iPSCs for transfection to achieve high nuclear delivery efficiency. When iPSCs are dissociated into single cells using a gentle-cell dissociation reagent and transfected using an optimized program, >50% cells can be induced to take up the large gene editing vectors. Because the AAVS1 locus is located in the intron of an active gene (PPP1R12C), a splicing acceptor (SA)-linked puromycin resistant gene (PAC) was used to select targeted iPSCs while excluding random integration-only and untransfected cells. This strategy greatly increases the chance of obtaining targeted clones, and can be used in other active gene targeting experiments as well. Two weeks after puromycin selection at the dose adjusted for the specific iPSC line, clones are ready to be picked by manual dissection of large, isolated colonies into smaller pieces that are transferred to fresh medium in a smaller well for further expansion and genetic and functional screening. One can follow this protocol to readily obtain multiple GFP reporter iPSC lines that are useful for in vivo and in vitro imaging and cell isolation.

TALEN-mediated gene editing at the safe harbor AAVS1 locus enables high-efficiency transgene addition in human iPSCs. This protocol describes the procedures for preparing iPSCs for TALEN and donor vector delivery, transfecting iPSCs, and selecting and isolating iPSC clones to achieve targeted integration of a GFP gene to generate reporter lines.

Targeted transgene addition can provide persistent gene expression while circumventing the gene silencing and insertional mutagenesis caused by viral ...

Developmental Biology

Constitutive and Inducible Systems for Genetic In Vivo Modification of Mouse Hepatocytes Using Hydrodynamic Tail Vein Injection

In research models of liver cancer, regeneration, inflammation, and fibrosis, flexible systems for in vivo gene expression and silencing are highly useful. Hydrodynamic tail vein injection of transposon-based constructs is an efficient method for genetic manipulation of hepatocytes in adult mice. In addition to constitutive transgene expression, this system can be used for more advanced applications, such as shRNA-mediated gene knock-down, implication of the CRISPR/Cas9 system to induce gene mutations, or inducible systems. Here, the combination of constitutive CreER expression together with inducible expression of a transgene or miR-shRNA of choice is presented as an example of this technique. We cover the multi-step procedure starting from the preparation of sleeping beauty-transposon constructs, to the injection and treatment of mice, and the preparation of liver tissue for analysis by immunostaining. The system presented is a reliable and efficient approach to achieve complex genetic manipulations in hepatocytes. It is specifically useful in combination with Cre/loxP-based mouse strains and can be applied to a variety of models in the research of liver disease.

Constitutive and Inducible Systems for Genetic In Vivo Modification of Mouse Hepatocytes Using Hydrodynamic Tail Vein Injection

Hydrodynamic tail vein injection of transposon-based integration vectors enables stable transfection of murine hepatocytes in vivo. Here, we present a practical protocol for transfection systems that enables the long-term constitutive expression of a single transgene or combined constitutive and doxycycline-inducible expression of a transgene or miR-shRNA in the liver.

In research models of liver cancer, regeneration, inflammation, and fibrosis, flexible systems for in vivo gene expression and silencing are highly ...

Genetics

CRISPR/Cas9-mediated Targeted Integration In Vivo Using a Homology-mediated End Joining-based Strategy

As a promising genome editing platform, the CRISPR/Cas9 system has great potential for efficient genetic manipulation, especially for targeted integration of transgenes. However, due to the low efficiency of homologous recombination (HR) and various indel mutations of non-homologous end joining (NHEJ)-based strategies in non-dividing cells, in vivo genome editing remains a great challenge. Here, we describe a homology-mediated end joining (HMEJ)-based CRISPR/Cas9 system for efficient in vivo precise targeted integration. In this system, the targeted genome and the donor vector containing homology arms (~800 bp) flanked by single guide RNA (sgRNA) target sequences are cleaved by CRISPR/Cas9. This HMEJ-based strategy achieves efficient transgene integration in mouse zygotes, as well as in hepatocytes in vivo. Moreover, a HMEJ-based strategy offers an efficient approach for correction of fumarylacetoacetate hydrolase (Fah) mutation in the hepatocytes and rescues Fah-deficiency induced liver failure mice. Taken together, focusing on targeted integration, this HMEJ-based strategy provides a promising tool for a variety of applications, including generation of genetically modified animal models and targeted gene therapies.

CRISPR/Cas9-mediated Targeted Integration In Vivo Using a Homology-mediated End Joining-based Strategy

The clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9 (CRISPR/Cas9) system provides a promising tool for genetic engineering, and opens up the possibility of targeted integration of transgenes. We describe a homology-mediated end joining (HMEJ)-based strategy for efficient DNA targeted integration in vivo and targeted gene therapies using CRISPR/Cas9.

As a promising genome editing platform, the CRISPR/Cas9 system has great potential for efficient genetic manipulation, especially for targeted integration ...

Genome Editing in Mammalian Cell Lines using CRISPR-Cas

The clustered regularly interspaced short palindromic repeats (CRISPR) system functions naturally in bacterial adaptive immunity, but has been successfully repurposed for genome engineering in many different living organisms. Most commonly, the wildtype CRISPR associated 9 (Cas9) or Cas12a endonuclease is used to cleave specific sites in the genome, after which the DNA double-stranded break is repaired via the non-homologous end joining (NHEJ) pathway or the homology-directed repair (HDR) pathway depending on whether a donor template is absent or present respectively. To date, CRISPR systems from different bacterial species have been shown to be capable of performing genome editing in mammalian cells. However, despite the apparent simplicity of the technology, multiple design parameters need to be considered, which often leave users perplexed about how best to carry out their genome editing experiments. Here, we describe a complete workflow from experimental design to identification of cell clones that carry desired DNA modifications, with the goal of facilitating successful execution of genome editing experiments in mammalian cell lines. We highlight key considerations for users to take note of, including the choice of CRISPR system, the spacer length, and the design of a single-stranded oligodeoxynucleotide (ssODN) donor template. We envision that this workflow will be useful for gene knockout studies, disease modeling efforts, or the generation of reporter cell lines.

CRISPR-Cas is a powerful technology to engineer the complex genomes of plants and animals. Here, we detail a protocol to efficiently edit the human genome using different Cas endonucleases. We highlight important considerations and design parameters to optimize editing efficiency.

The clustered regularly interspaced short palindromic repeats (CRISPR) system functions naturally in bacterial adaptive immunity, but has been ...

Introducing Point Mutations into Human Pluripotent Stem Cells Using Seamless Genome Editing

Custom designed endonucleases, such as RNA-guided Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9, enable efficient genome editing in mammalian cells. Here we describe detailed procedures to seamlessly genome edit the hepatocyte nuclear factor 4 alpha (HNF4&#945;) locus as an example in human pluripotent stem cells. Combining a piggyBac-based donor plasmid and the CRISPR-Cas9 nickase mutant in a two-step genetic selection, we demonstrate correct and efficient targeting of the HNF4&#945; locus.

Here, we describe a detailed method for seamless gene editing in human pluripotent stem cells using a piggyBac-based donor plasmid and the Cas9 nickase mutant. Two point mutations were introduced into exon 8 of the hepatocyte nuclear factor 4 alpha (HNF4&#945;) locus in human embryonic stem cells (hESCs).

Custom designed endonucleases, such as RNA-guided Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9, enable efficient genome editing ...

Rapid Assembly of Multi-Gene Constructs using Modular Golden Gate Cloning

The Golden Gate cloning method enables the rapid assembly of multiple genes in any user-defined arrangement. It utilizes type IIS restriction enzymes that cut outside of their recognition sites and create a short overhang. This modular cloning (MoClo) system uses a hierarchical workflow in which different DNA parts, such as promoters, coding sequences (CDS), and terminators, are first cloned into an entry vector. Multiple entry vectors then assemble into transcription units. Several transcription units then connect into a multi-gene plasmid. The Golden Gate cloning strategy is of tremendous advantage because it allows scar-less, directional, and modular assembly in a one-pot reaction. The hierarchical workflow typically enables the facile cloning of a large variety of multi-gene constructs with no need for sequencing beyond entry vectors. The use of fluorescent protein dropouts enables easy visual screening. This work provides a detailed, step-by-step protocol for assembling multi-gene plasmids using the yeast modular cloning (MoClo) kit. We show optimal and suboptimal results of multi-gene plasmid assembly and provide a guide for screening for colonies. This cloning strategy is highly applicable for yeast metabolic engineering and other situations in which multi-gene plasmid cloning is required.

The goal of this protocol is to provide a detailed, step-by-step guide for assembling multi-gene constructs using the modular cloning system based on Golden Gate cloning. It also gives recommendations on critical steps to ensure optimal assembly based on our experiences.

The Golden Gate cloning method enables the rapid assembly of multiple genes in any user-defined arrangement. It utilizes type IIS restriction enzymes that ...

Bioengineering

Gene Knock-in by CRISPR/Cas9 and Cell Sorting in Macrophage and T Cell Lines

Functional genomics studies of the immune system require genetic manipulations that involve both deletion of target genes and addition of elements to proteins of interest. Identification of gene functions in cell line models is important for gene discovery and exploration of cell-intrinsic mechanisms. However, genetic manipulations of immune cells such as T cells and macrophage cell lines using CRISPR/Cas9-mediated knock-in are difficult because of the low transfection efficiency of these cells, especially in a quiescent state. To modify genes in immune cells, drug-resistance selection and viral vectors are typically used to enrich for cells expressing the CRIPSR/Cas9 system, which inevitably results in undesirable intervention of the cells. In a previous study, we designed dual fluorescent reporters coupled to CRISPR/Cas9 that were transiently expressed after electroporation. This technical solution leads to rapid gene deletion in immune cells; however, gene knock-in in immune cells such as T cells and macrophages without the use of drug-resistance selection or viral vectors is even more challenging. In this article, we show that by using cell sorting to aid selection of cells transiently expressing CRISPR/Cas9 constructs targeting the Rosa26 locus in combination with a donor plasmid, gene knock-in can be achieved in both T cells and macrophages without drug-resistance enrichment. As an example, we show how to express human ACE2, a receptor of SARS-Cov-2, which is responsible for the current Covid-19 pandemic, in RAW264.7 macrophages by performing knock-in experiments. Such gene knock-in cells can be widely used for mechanistic studies.

This protocol uses fluorescent reporters and cell sorting to simplify knock-in experiments in macrophage and T cell lines. Two plasmids are used for these simplified knock-in experiments, namely a CRISPR/Cas9- and DsRed2-expressing plasmid and a homologous recombination donor plasmid expressing EBFP2, which is permanently integrated at the Rosa26 locus in immune cells.

Functional genomics studies of the immune system require genetic manipulations that involve both deletion of target genes and addition of elements to ...

Immunology and Infection

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

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

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

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

Crude Vector Production and Efficient DNA Delivery in Cell Cultures

Transgene Expression in Cultured Cells Using Unpurified Recombinant Adeno-Associated Viral Vectors

Recombinant adeno-associated viral vectors (rAAV) can achieve potent and durable transgene expression without integration in a broad range of tissue types, making them a popular choice for gene delivery in animal models and in clinical settings. In addition to therapeutic applications, rAAVs are a useful laboratory tool for delivering transgenes tailored to the researcher&#39;s experimental needs and scientific goals in cultured cells. Some examples include exogenous reporter genes, overexpression cassettes, RNA interference, and CRISPR-based tools, including those for genome-wide screens. rAAV transductions are less harmful to cells than electroporation or chemical transfection and do not require any special equipment or expensive reagents to produce. Crude lysates or conditioned media containing rAAVs can be added directly to cultured cells without further purification to transduce many cell types&#8212;an underappreciated feature of rAAVs. Here, we provide protocols for basic transgene cassette cloning and demonstrate how to produce and apply crude rAAV preparations to cultured cells. As proof of principle, we demonstrate the transduction of three cell types that have not yet been reported in rAAV applications: placental cells, myoblasts, and small intestinal organoids. We discuss appropriate uses for crude rAAV preparations, the limitations of rAAVs for gene delivery, and considerations for capsid choice. This protocol outlines a simple, low-cost, and effective method for researchers to achieve productive DNA delivery in cell culture using rAAV without the need for laborious titration and purification steps.

Author Spotlight: Unveiling the Potential of Unpurified Recombinant AAVs in Cell Culture Research 

Recombinant adeno-associated virus (rAAV) is widely used for clinical and preclinical gene delivery. An underappreciated use for rAAVs is the robust transduction of cultured cells without the need for purification. For researchers new to rAAV, we provide a protocol for transgene cassette cloning, crude vector production, and cell culture transduction.

Recombinant adeno-associated viral vectors (rAAV) can achieve potent and durable transgene expression without integration in a broad range of tissue ...

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

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

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

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

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

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

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

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