We thank Stephan Wilkens for help in transmission electron microscopy. This work was supported by the National Institutes of Health Grant R01AG023731.

Previous studies have shown that the first amino-terminal 22 residues of Mgm101 are cleaved after import into mitochondria 19. For expression in Escherichia coli, the MGM101 open reading frame lacking the first 22 codons is amplified by PCR and placed downstream of the malE sequence encoding the maltose binding protein (MBP) in a modified version of the expression vector pMAL-c2E. This generates the MBP-Mgm101 fusion with a linker containing a cleavage site for the PreScission protease (Figure 3). The plasmid is first constructed by selecting E. coli transformants without the IPTG and Xgal blue/white selection. The resulting plasmid pMAL-c2E-MGM101 is then introduced into the E. coli strain BL21-CodonPlus(DE3)-RIL by selecting ampicillin and chloramphenicol resistant colonies.
1. Expression, Induction, Cell Lysis and DNase I Treatment
<ol>
	<li>Inoculate fresh transformants in 10 ml of LB medium (1% Bacto tryptone, 0.5% yeast extract, 1% NaCl) supplemented with glucose (0.2%), ampicillin (100 &mu;g/ml) and chloramphenicol (50 &mu;g/ml). Incubate at 37 &deg;C O/N with shaking at 200 rpm.</li>
	<li>Inoculate the 10 ml preculture into 2 liters of the supplemented LB medium as above. Grow the cells at 37 &deg;C with shaking until OD600 reaches 0.5.</li>
	<li>Induce expression of the MBP-Mgm101 fusion protein by adding Isopropyl &beta;-D-1-thiogalactopyranoside (IPTG) at a final concentration of 0.3 mM. Grow the cells with shaking at 30 &deg;C for 5 hr.</li>
	<li>Collect the cells by centrifugation using a Beckman JA-10 rotor (5,500 x g, 4 &deg;C, 10 min). After discarding the supernatant, resuspend the cell pellet in 30 ml of lysis buffer (20 mM Tris-HCl, pH 7.4 and 1 mM EDTA, pH 7.4) containing protease inhibitors (25 &mu;M leupeptin, 1 &mu;M pepstatin A and 1 mM PMSF).</li>
	<li>Sonicate cell suspension on ice for 20 sec using an ultrasonic processor (Heat Systems; Model W-385) at 50% duty cycle, allow to cool on ice for 30 sec, and repeat 2x.</li>
	<li>Add 1 ml of DNAse 1 stock at 2 mg/ml. Rock the cell lysate at 4 &deg;C for 2 hr.</li>
	<li>Adjust NaCl to a final concentration of 500 mM.</li>
	<li>Remove the cell debris by centrifugation at 10,000 x g at 4 &deg;C for 30 min.</li>
</ol>
2. Purification with Amylose Affinity Chromatography
<ol>
	<li>Equilibrate 1.5 ml of amylose resin (50% slurry) in the lysis buffer. Add the equilibrated amylose resin to the cell lysate. Rock gently at 4 &deg;C O/N.</li>
	<li>Load the lysate-resin mix onto an Econo-Column chromatography column installed in a cold room. Allow the unbound proteins to pass the column by gravity.</li>
	<li>Wash the amylose resin with 300 ml of the wash buffer I (20 mM Tris-HCl, pH 7.4; 400 mM NaCl; 1 mM EDTA, pH 7.4; and 0.2 mM PMSF).</li>
	<li>Repeat the wash with 150 ml of wash buffer II (20 mM Tris-HCl, pH 7.4; 200 mM NaCl, 1 mM EDTA, pH 7.4; and 0.2 mM PMSF).</li>
	<li>Add 5 ml of elution buffer (20 mM Tris-HCl, pH 7.4; 200 mM NaCl; 1 mM EDTA, pH 7.4; and 0.2 mM PMSF, 10 mM maltose) to the column, incubate at 4 &deg;C for 10 min, collect the eluate and repeat for more times.</li>
	<li>Combine the eluates, determine the yield and purity of MBP-Mgm101 by loading an aliquot of the eluate on a SDS-PAGE gel followed by coomassie staining (Figure 4).</li>
</ol>
3. PreScission Protease Cleavage and Cation Exchange Chromatography
<ol>
	<li>Add 50 units of PreScission protease to the MBP-Mgm101 eluate. Perform the cleavage at 4 &deg;C O/N and allow it to continue during dialysis against the dialysis buffer (20 mM Tris-HCl, pH 7.4; 100 mM NaCl; 2 mM DTT; 1 mM EDTA, pH 7.4; and 0.2 mM PMSF)</li>
	<li>Check the efficiency of cleavage on a SDS-PAGE gel (Figure 5A).</li>
	<li>Load the cleaved MBP-Mgm101 by multiple injections on a Bio-Scale Mini Macro-Prep High S cartridge connected to a Bio-Rad Biologic DuoFlow FPLC system.</li>
	<li>Start the cation exchange chromatography by applying a step salt gradient of 5 mM, 300 mM, 500 mM, 750 mM and 1,000 mM of NaCl that is created by mixing Buffer A (5 mM NaCl; 10 mM Na-phosphate, pH 7.2; 1 mM PMSF) and Buffer B (1 M NaCl; 10 mM Na-phosphate, pH 7.2; 1 mM PMSF). Set the flow rate at 0.5 ml/min. Collect the Mgm101 fraction and check the purity of the protein on a SDS-PAGE gel (Figure 5B).</li>
</ol>
4. Size Exclusion Chromatography
<ol>
	<li>Combine the protein fractions from cation exchange chromatography. Dialyze the protein in GF equilibration buffer (20 mM MOPS, pH 7.0; 150 mM NaCl; 1mM EDTA, pH 7.4; 5 mM 2-Mercaptoethanol; 0.2 mM PMSF).</li>
	<li>Concentrate the protein with VIVASPIN 15R Ultrafiltration spin column to reduce the volume to ~1 ml by spinning at 3,000 x g at 4 &deg;C.</li>
	<li>Load Mgm101 on a calibrated Superose 6 prep grade column equilibrated with the chromatography buffer (20 mM MOPS, pH 7.0; 150 mM NaCl; 5 mM &beta;-mercaptoethanol; 1 mM EDTA, pH 7.4; 0.2 mM PMSF).</li>
	<li>Start the size exclusion with the chromatography buffer run at a flow rate of 0.5 ml/min.</li>
	<li>Collect the fractions of the purified Mgm101 peaks. Load aliquots of the fractions on a 12% SDS-PAGE for checking the final quality of the protein.</li>
	<li>Concentrate the protein with VIVASPIN 6, then quickly freeze the samples in liquid N2 and store at -80 &deg;C.</li>
	<li>Use the Mgm101 samples within 6-12 months for ssDNA-binding assay (Figure 8) and for structural visualization by transmission electron microscopy (Figure 9).</li>
</ol>

<ol>
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K., Park, D., Xu, L., Kleckner, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DMC1:+a+meiosis-specific+yeast+homolog+of+E.+coli+recA+required+for+recombination,+synaptonemal+complex+formation,+and+cell+cycle+progression.">DMC1: a meiosis-specific yeast homolog of E. coli recA required for recombination, synaptonemal complex formation, and cell cycle progression.</a> Cell. 69, 439-456 (1992).</li><li>Shinohara, A., Ogawa, H., Ogawa, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rad51+protein+involved+in+repair+and+recombination+in+S.+cerevisiae+is+a+RecA-like+protein.">Rad51 protein involved in repair and recombination in S. cerevisiae is a RecA-like protein.</a> Cell. 69, 457-470 (1992).</li><li>Passy, S. 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U.S.A. 96, 4279-4284 (1999).</li><li>Ploquin, M., 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=Functional+and+structural+basis+for+a+bacteriophage+homolog+of+human+RAD52.">Functional and structural basis for a bacteriophage homolog of human RAD52.</a> Curr. Biol. 18, 1142-1146 (2008).</li><li>Chen, X. J., Guan, M. X., Clark-Walker, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MGM101,+a+nuclear+gene+involved+in+maintenance+of+the+mitochondrial+genome+in+Saccharomyces+cerevisiae.">MGM101, a nuclear gene involved in maintenance of the mitochondrial genome in Saccharomyces cerevisiae.</a> Nucl. 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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+properly+configured+ring+structure+is+critical+for+the+function+of+the+mitochondrial+DNA+recombination+protein.">A properly configured ring structure is critical for the function of the mitochondrial DNA recombination protein.</a> Mgm101. J. Biol. Chem. 287, 37259-37268 (2012).</li><li>Zuo, X., Xue, D., Li, N., Clark-Walker, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+functional+core+of+the+mitochondrial+genome+maintenance+protein+Mgm101p+in+Saccharomyces+cerevisiae+determined+with+a+temperature-conditional+allele.">A functional core of the mitochondrial genome maintenance protein Mgm101p in Saccharomyces cerevisiae determined with a temperature-conditional allele.</a> FEMS Yeast Res. 7, 131-140 (2007).</li><li>Itoh, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+packaging+proteins+Glom+and+Glom2+coordinately+organize+the+mitochondrial+nucleoid+of+Physarum+polycephalum.">DNA packaging proteins Glom and Glom2 coordinately organize the mitochondrial nucleoid of Physarum polycephalum.</a> Mitochondrion. 11, 575-586 (2011).</li><li>Janicka, S., 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=A+RAD52-like+single-stranded+DNA+binding+protein+affects+mitochondrial+DNA+repair+by+recombination.">A RAD52-like single-stranded DNA binding protein affects mitochondrial DNA repair by recombination.</a> Plant J. 72, 423-435 (2012).</li></ol>

The authors declare that they have no competing financial interests.

It has been a challenge to produce a stable, native recombinant Mgm101 protein from E. coli possibly due to its insolubility in bacterial cells. In this report, we show that the MBP-fusion strategy allows the protein to be expressed at a reasonably high level. By using negative staining transmission electron microscopy and size exclusion chromatography, we have previously shown that the MBP-fusion protein forms uniform oligomers in vitro 18. It is possible that the folding and oligomerization...

Homologous recombination is important for the repair of double-strand breaks (DSBs) and interstrand crosslinks, and for the reinitiation of DNA replication from collapsed replication forks 1. In conventional homologous recombination, the central reaction is catalyzed by the ATP-dependent recombinases including RecA in prokaryotes, and Rad51 and Dmc1 in eukaryotes 1-3. These recombinases form nucleoprotein filaments on ssDNA, which are essential for initiating homology search and strand invasion within duplex DNA templates (Figure 1, left panel) 4-7. In addition to the conventional scheme, homologous recombination can also take place in a RecA/Rad51-independent manner (Figure 1, right panel). For instance, the yeast Rad52 and Rad59 proteins can directly catalyze the annealing of complementary ssDNA strands which are exposed by resectioning of dsDNA breaks. This recombination process, known as single strand annealing, generally does not involve homologous pairing with dsDNA templates. After annealing, heterologous tails are removed by exonucleases and nicks are ligated to restore genome continuity 8-10. Repair by the single strand annealing mechanism is often accompanied by deletions of genomic sequences between directly repeated regions.
Rad52 belongs to a diverse group of recombination proteins that are widespread among bacteriophages 11. These proteins are also known as Single Strand Annealing Proteins (SSAPs), based on their activity in promoting the annealing of homologous single stranded DNA molecules. The best characterized bacteriophage SSAPs are Red&beta; and Erf from the bacteriophages &lambda; and P22, RecT from the prophage rac and the Sak protein from the lactococcal phage ul36. The SSAPs are structurally characterized by a typical &beta;-&beta;-&beta;-&alpha; fold, although similarity is virtually undetectable in their primary sequences. They all form large homo-oligomeric rings of 10 - 14 fold symmetry in vitro 12-14. The functional implications of this characteristic higher order structural organization is not well understood.
We are interested in understanding the mechanism of homologous recombination in the mitochondrial genome. We have previously identified the MGM101 gene that is essential for the maintenance of mtDNA in Saccharomyces cerevisiae 15. MGM101 was subsequently found to be associated with mitochondrial nucleoids and is required for the tolerance of mtDNA to DNA-damaging agents 16. However, the study of Mgm101 has been held back in the last decade by the difficulty to produce recombinant Mgm101. We have recently succeeded in producing soluble Mgm101 at large quantities from E. coli using the MBP-fusion strategy. This has enabled us to demonstrate that Mgm101 shares biochemical and structural similarities with the Rad52-family of proteins 17,18. In this report, a three-step purification procedure is described, which produces homogeneous Mgm101for biochemical and structural analyses (Figure 2).

<table><tbody><tr><td>Expression vector pMAL-c2E</td><td>New England Biolabs</td><td>#N8066S</td><td></td></tr><tr><td>PreScission Protease</td><td>GE Healthcare Life Sciences</td><td>#27-0843-01</td><td></td></tr><tr><td>BL21-CodonPlus(DE3)-RIL cells</td><td>Strategene</td><td>#230245</td><td></td></tr><tr><td>Leupeptin</td><td>Roche Applied Science</td><td>#11034626001</td><td></td></tr><tr><td>Pepstatin</td><td>Roche Applied Science</td><td>#11359053001</td><td></td></tr><tr><td>Phenylmethylsulfonyl fluoride (PMSF)</td><td>Roche Applied Science</td><td>#10837091001</td><td></td></tr><tr><td>DNAse I</td><td>Sigma</td><td>#DN25-1G</td><td></td></tr><tr><td>Isopropyl &amp;#946;-D-1-thiogalactopyranoside (IPTG)</td><td>Roche Applied Science</td><td>#11411446001</td><td></td></tr><tr><td>Amylose resin</td><td>New England Biolabs</td><td>#E8021L</td><td></td></tr><tr><td>Econo-Column chromatography column</td><td>BIO-RAD</td><td>#7372512</td><td></td></tr><tr><td>Bio-Scale Mini Macro-Prep High S cartridge (1 ml)</td><td>BIO-RAD</td><td>#732-4130</td><td></td></tr><tr><td>VIVASPIN 15R Ultrafiltration spin column (10,000 MWCO)</td><td>Sartorius Stedium</td><td>#VS15RH02</td><td></td></tr><tr><td>Superose 6 prep grade column</td><td>Amersham Bioscirnces</td><td>#17-0489-01</td><td></td></tr><tr><td>VIVASPIN 6 Ultrafiltration spin column (5,000 MWCO)</td><td>Sartorius Stedium</td><td>#VS0611</td><td></td></tr></tbody></table>

preparation of the mgm101 recombination protein by mbp-based tagging strategy

The MGM101 gene was identified 20 years ago for its role in the maintenance of mitochondrial DNA. Studies from several groups have suggested that the Mgm101 protein is involved in the recombinational repair of mitochondrial DNA. Recent investigations have indicated that Mgm101 is related to the Rad52-type recombination protein family. These proteins form large oligomeric rings and promote the annealing of homologous single stranded DNA molecules. However, the characterization of Mgm101 has been hindered by the difficulty in producing the recombinant protein. Here, a reliable procedure for the preparation of recombinant Mgm101 is described. Maltose Binding Protein (MBP)-tagged Mgm101 is first expressed in Escherichia coli. The fusion protein is initially purified by amylose affinity chromatography. After being released by proteolytic cleavage, Mgm101 is separated from MBP by cationic exchange chromatography. Monodispersed Mgm101 is then obtained by size exclusion chromatography. A yield of ~0.87 mg of Mgm101 per liter of bacterial culture can be routinely obtained. The recombinant Mgm101 has minimal contamination of DNA. The prepared samples are successfully used for biochemical, structural and single particle image analyses of Mgm101. This protocol may also be used for the preparation of other large oligomeric DNA-binding proteins that may be misfolded and toxic to bacterial cells.

Mgm101 is a Rad52-related recombination protein in mitochondria. Rad52 has been extensively studied for its role in mitochondrial DNA recombination (Figure 1). Recombinant Mgm101 can be prepared by a three-step procedure (Figure 2). This is facilitated by the use of the MBP-tagging strategy that allows Mgm101 to be expressed in a soluble form and subsequently released from the tag by proteolytic cleavage (Figure 3).
In a typical preparation, a...

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Preparation of the Mgm101 Recombination Protein by MBP-based Tagging Strategy

The yeast mitochondrial nucleoid protein, Mgm101, is a Rad52-type recombination protein that forms large oligomeric rings. A protocol is described to prepare soluble recombinant Mgm101 using the Maltose Binding Protein (MBP)-tagging strategy coupled with cation exchange and size exclusion chromatography.

The MGM101 gene was identified 20 years ago for its role in the maintenance of mitochondrial DNA. Studies from several groups have suggested that the ...

preparation-mgm101-recombination-protein-mbp-based-tagging

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12.0K Views.  State University of New York Upstate Medical University. The yeast mitochondrial nucleoid protein, Mgm101, is a Rad52-type recombination protein that forms large oligomeric rings. A protocol is described to prepare soluble recombinant Mgm101 using the Maltose Binding Protein (MBP)-tagging strategy coupled with cation exchange and size exclusion chromatography.

Video: Preparation of the Mgm101 Recombination Protein by MBP-based Tagging Strategy

Engineering Artificial Factors to Specifically Manipulate Alternative Splicing in Human Cells

The processing of most eukaryotic RNAs is mediated by RNA Binding Proteins (RBPs) with modular configurations, including an RNA recognition module, which specifically binds the pre-mRNA target and an effector domain. Previously, we have taken advantage of the unique RNA binding mode of the PUF domain in human Pumilio 1 to generate a programmable RNA binding scaffold, which was used to engineer various artificial RBPs to manipulate RNA metabolism. Here, a detailed protocol is described to construct Engineered Splicing Factors (ESFs) that are specifically designed to modulate the alternative splicing of target genes. The protocol includes how to design and construct a customized PUF scaffold for a specific RNA target, how to construct an ESF expression plasmid by fusing a designer PUF domain and an effector domain, and how to use ESFs to manipulate the splicing of target genes. In the representative results of this method, we have also described the common assays of ESF activities using splicing reporters, the application of ESF in cultured human cells, and the subsequent effect of splicing changes. By following the detailed protocols in this report, it is possible to design and generate ESFs for the regulation of different types of Alternative Splicing (AS), providing a new strategy to study splicing regulation and the function of different splicing isoforms. Moreover, by fusing different functional domains with a designed PUF domain, researchers can engineer artificial factors that target specific RNAs to manipulate various steps of RNA processing.

This report describes a bioengineering method to design and construct novel Artificial Splicing Factors (ASFs) that specifically modulate the splicing of target genes in mammalian cells. This method can be further expanded to engineer various artificial factors to manipulate other aspects of RNA metabolism.

The processing of most eukaryotic RNAs is mediated by RNA Binding Proteins (RBPs) with modular configurations, including an RNA recognition module, which ...

Genetics

Inducible T7 RNA Polymerase-mediated Multigene Expression System, pMGX

Co-expression of multiple proteins is increasingly essential for synthetic biology, studying protein-protein complexes, and characterizing and harnessing biosynthetic pathways. In this manuscript, the use of a highly effective system for the construction of multigene synthetic operons under the control of an inducible T7 RNA polymerase is described. This system allows many genes to be expressed simultaneously from one plasmid. Here, a set of four related vectors, pMGX-A, pMGX-hisA, pMGX-K, and pMGX-hisK, with either the ampicillin or kanamycin resistance selectable marker (A and K) and either possessing or lacking an N-terminal hexahistidine tag (his) are disclosed. Detailed protocols for the construction of synthetic operons using this vector system are provided along with the corresponding data, showing that a pMGX-based system containing five genes can be readily constructed and used to produce all five encoded proteins in Escherichia coli. This system and protocol enables researchers to routinely express complex multi-component modules and pathways in E. coli.

This study describes methods for the T7-mediated co-expression of multiple genes from a single plasmid in Escherichia coli using the pMGX plasmid system.

Co-expression of multiple proteins is increasingly essential for synthetic biology, studying protein-protein complexes, and characterizing and harnessing ...

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 ...

Genetic Manipulation in &Delta;ku80 Strains for Functional Genomic Analysis of Toxoplasma gondii

Targeted genetic manipulation using homologous recombination is the method of choice for functional genomic analysis to obtain a detailed view of gene function and phenotype(s). The development of mutant strains with targeted gene deletions, targeted mutations, complemented gene function, and/or tagged genes provides powerful strategies to address gene function, particularly if these genetic manipulations can be efficiently targeted to the gene locus of interest using integration mediated by double cross over homologous recombination.
Due to very high rates of nonhomologous recombination, functional genomic analysis of Toxoplasma gondii has been previously limited by the absence of efficient methods for targeting gene deletions and gene replacements to specific genetic loci. Recently, we abolished the major pathway of nonhomologous recombination in type I and type II strains of T. gondii by deleting the gene encoding the KU80 protein1,2. The &Delta;ku80 strains behave normally during tachyzoite (acute) and bradyzoite (chronic) stages in vitro and in vivo and exhibit essentially a 100% frequency of homologous recombination. The &Delta;ku80 strains make functional genomic studies feasible on the single gene as well as on the genome scale1-4.
Here, we report methods for using type I and type II &Delta;ku80&Delta;hxgprt strains to advance gene targeting approaches in T. gondii. We outline efficient methods for generating gene deletions, gene replacements, and tagged genes by targeted insertion or deletion of the hypoxanthine-xanthine-guanine phosphoribosyltransferase (HXGPRT) selectable marker. The described gene targeting protocol can be used in a variety of ways in &Delta;ku80 strains to advance functional analysis of the parasite genome and to develop single strains that carry multiple targeted genetic manipulations. The application of this genetic method and subsequent phenotypic assays will reveal fundamental and unique aspects of the biology of T. gondii and related significant human pathogens that cause malaria (Plasmodium sp.) and cryptosporidiosis (Cryptosporidium).

Here we report a method for using type I and type II &Delta;ku80 strains of Toxoplasma gondii to efficiently generate targeted gene deletions and gene replacements for functional genomic analysis.

Targeted genetic manipulation using homologous recombination is the method of choice for functional genomic analysis to obtain a detailed view of gene ...

Immunology and Infection

Zebrafish Model of Neuroblastoma Metastasis

Zebrafish has emerged as an important animal model to study human diseases, especially cancer. Along with the robust transgenic and genome editing technologies applied in zebrafish modeling, the ease of maintenance, high-yield productivity, and powerful live imaging altogether make the zebrafish a valuable model system to study metastasis and cellular and molecular bases underlying this process in vivo. The first zebrafish neuroblastoma (NB) model of metastasis was developed by overexpressing two oncogenes, MYCN and LMO1, under control of the dopamine-beta-hydroxylase (d&#946;h) promoter. Co-overexpressed MYCN and LMO1 led to the reduced latency and increased penetrance of neuroblastomagenesis, as well as accelerated distant metastasis of tumor cells. This new model reliably reiterates many key features of human metastatic NB, including involvement of clinically relevant and metastasis-associated genetic alterations; natural and spontaneous development of metastasis in vivo; and conserved sites of metastases. Therefore, the zebrafish model possesses unique advantages to dissect the complex process of tumor metastasis in vivo.

This paper introduces the method of developing, characterizing, and tracking in real-time the tumor metastasis in zebrafish model of neuroblastoma, specifically in the transgenic zebrafish line with overexpression of MYCN and LMO1, which develops metastasis spontaneously.

Zebrafish has emerged as an important animal model to study human diseases, especially cancer. Along with the robust transgenic and genome editing ...

Cancer Research

Photoactivated Localization Microscopy with Bimolecular Fluorescence Complementation (BiFC-PALM)

Protein-protein interactions (PPIs) are key molecular events to biology. However, it remains a challenge to visualize PPIs with sufficient resolution and sensitivity in cells because the resolution of conventional light microscopy is diffraction-limited to ~250 nm. By combining bimolecular fluorescence complementation (BiFC) with photoactivated localization microscopy (PALM), PPIs can be visualized in cells with single molecule sensitivity and nanometer spatial resolution. BiFC is a commonly used technique for visualizing PPIs with fluorescence contrast, which involves splitting of a fluorescent protein into two non-fluorescent fragments. PALM is a recent superresolution microscopy technique for imaging biological samples at the nanometer and single molecule scales, which uses phototransformable fluorescent probes such as photoactivatable fluorescent proteins (PA-FPs). BiFC-PALM was demonstrated by splitting PAmCherry1, a PA-FP compatible with PALM, for its monomeric nature, good single molecule brightness, high contrast ratio, and utility for stoichiometry measurements. When split between amino acids 159 and 160, PAmCherry1 can be made into a BiFC probe that reconstitutes efficiently at 37 &#176;C with high specificity to PPIs and low non-specific reconstitution. Ras-Raf interaction is used as an example to show how BiFC-PALM helps to probe interactions at the nanometer scale and with single molecule resolution. Their diffusion can also be tracked in live cells using single molecule tracking (smt-) PALM. In this protocol, factors to consider when designing the fusion proteins for BiFC-PALM are discussed, sample preparation, image acquisition, and data analysis steps are explained, and a few exemplary results are showcased. Providing high spatial resolution, specificity, and sensitivity, BiFC-PALM is a useful tool for studying PPIs in intact biological samples.

Photoactivated Localization Microscopy with Bimolecular Fluorescence Complementation BiFC-PALM

Protein-protein interactions are visualized in cells with nanometer spatial resolution by combining bimolecular fluorescence complementation (BiFC) with photoactivated localization microscopy (PALM). Described here is the use of BiFC-PALM for imaging Ras-Raf interactions in U2OS cells for visualizing the nanoscale clustering and diffusion of individual Ras-Raf complexes.

Protein-protein interactions (PPIs) are key molecular events to biology. However, it remains a challenge to visualize PPIs with sufficient resolution and ...

Bioengineering

Enrichment of Native and Recombinant Extracellular Vesicles of Mycobacteria

Most bacteria, including mycobacteria, generate extracellular vesicles (EVs). Since bacterial EVs (bEVs) contain a subset of cellular components, including metabolites, lipids, proteins, and nucleic acids, several groups have evaluated either the native or recombinant versions of bEVs for their protective potency as subunit vaccine candidates. Unlike native EVs, recombinant EVs are molecularly engineered to contain one or more immunogens of interest. Over the last decade, different groups have explored diverse approaches for generating recombinant bEVs. However, here, we report the design, construction, and enrichment of recombinant mycobacterial EVs (mEVs) in mycobacteria. Towards that, we use Mycobacterium smegmatis (Msm), an avirulent soil mycobacterium as the model system. We first describe the generation and enrichment of native EVs of Msm. Then, we describe the design and construction of recombinant mEVs that contain either mCherry, a red fluorescent reporter protein, or EsxA (Esat-6), a prominent immunogen of Mycobacterium tuberculosis. We achieve this by separately fusing mCherry and EsxA N-termini with the C-terminus of a small Msm protein Cfp-29. Cfp-29 is one of the few abundantly present proteins of MsmEVs. The protocol to generate and enrich recombinant mEVs from Msm remains identical to the generation and enrichment of native EVs of Msm.

This protocol details the enrichment of native mycobacterial extracellular vesicles (mEVs) from axenic cultures of Mycobacterium smegmatis (Msm) and how mCherry (a red fluorescent reporter)-containing recombinant MsmEVs can be designed and enriched. Lastly, it verifies the novel approach with the enrichment of MsmEVs containing the EsxA protein of Mycobacterium tuberculosis.

Most bacteria, including mycobacteria, generate extracellular vesicles (EVs). Since bacterial EVs (bEVs) contain a subset of cellular components, ...

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

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

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

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

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

One-step CRISPR-based Strategy for Endogenous Gene Tagging in Drosophila melanogaster

The study of protein subcellular localization, dynamics, and regulation in live cells has been profoundly transformed by the advent of techniques that allow the tagging of endogenous genes to produce fluorescent fusion proteins. These methods enable researchers to visualize protein behavior in real time, providing valuable insights into their functions and interactions within the cellular environment. Many current gene tagging studies employ a two-step process where visible markers, such as eye color changes, are used to identify genetically modified organisms in the first step, and the visible marker is excised in the second step. Here, we present a one-step protocol to perform precise and rapid endogenous gene tagging in Drosophila melanogaster, which enables screening for engineered lines without the visible eye marker, offering a significant advantage over past methods. To screen for successful gene-tagging events, we employ a PCR-based technique to genotype individual flies by analyzing a small segment from their middle leg. Flies that pass the screening criteria are then used to produce stable stocks. Here, we detail the design and construction of CRISPR editing plasmids and methods for screening and confirmation of engineered lines. Together, this protocol improves the efficiency of endogenous gene tagging in Drosophila significantly and enables studies of cellular processes in vivo.

One-step CRISPR-based Strategy for Endogenous Gene Tagging in Drosophila melanogaster

Here, we present a simplified endogenous gene tagging protocol for Drosophila, which utilizes a PCR-based technique for marker-free identification of successful genetic modifications, facilitating the development of stable knock-in lines.

The study of protein subcellular localization, dynamics, and regulation in live cells has been profoundly transformed by the advent of techniques that ...

Neuroscience

Genetic Modification of Cyanobacteria by Conjugation Using the CyanoGate Modular Cloning Toolkit

Cyanobacteria are a diverse group of prokaryotic photosynthetic organisms that can be genetically modified for the renewable production of useful industrial commodities. Recent advances in synthetic biology have led to development of several cloning toolkits such as CyanoGate, a standardized modular cloning system for building plasmid vectors for subsequent transformation or conjugal transfer into cyanobacteria. Here we outline a detailed method for assembling a self-replicating vector (e.g., carrying a fluorescent marker expression cassette) and conjugal transfer of the vector into the cyanobacterial strains Synechocystis sp. PCC 6803 or Synechococcus elongatus UTEX 2973. In addition, we outline how to characterize the performance of a genetic part (e.g., a promoter) using a plate reader or flow cytometry.

Here, we present a protocol describing how to i) assemble a self-replicating vector using the CyanoGate modular cloning toolkit, ii) introduce the vector into a cyanobacterial host by conjugation, and iii) characterize transgenic cyanobacteria strains using a plate reader or flow cytometry.

Preparation of the Mgm101 Recombination Protein by MBP-based Tagging Strategy

Summary

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Chapters in this video

Engineering Artificial Factors to Specifically Manipulate Alternative Splicing in Human Cells

Inducible T7 RNA Polymerase-mediated Multigene Expression System, pMGX

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

Genetic Manipulation in Δku80 Strains for Functional Genomic Analysis of Toxoplasma gondii

Zebrafish Model of Neuroblastoma Metastasis

Photoactivated Localization Microscopy with Bimolecular Fluorescence Complementation (BiFC-PALM)

Enrichment of Native and Recombinant Extracellular Vesicles of Mycobacteria

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

One-step CRISPR-based Strategy for Endogenous Gene Tagging in Drosophila melanogaster

Genetic Modification of Cyanobacteria by Conjugation Using the CyanoGate Modular Cloning Toolkit