Click Chemistry: What it means for biologists

A few months ago I read a very nice blog post from our friends at TriLink Biotechnologies giving the chemist’s perspective on the excitement surrounding “Click Chemistry” and how it can be used to make non-natural, yet functional DNA and RNA. Some of the terms in that post such as 1,3-dipolar cycloaddition are oriented more towards chemists. Here’s a more biologist-friendly explanation of Click Chemistry: [Read more…]

PEI transfection patents to expire

July 7, 2015 marked the 20-year anniversary of the filing dates of both the U.S. patent and European patent limiting the use of PolyEthylenImine (PEI) as a transfection reagent. Coincidentally, both U.S. and European patents generally have a term of 20 years from the filing date.

Many academic researchers have been ignoring these patents and/or have been sharing protocols online and publishing articles explaining how cost-effective and simple PEI-mediated transfection can be. For example, the most commonly used PEI, catalog nr. 07923966-2 (Polysciences), is a linear form with molecular weight of 25,000 Da. The 2 gram bottle of PEI powder can be used to make a few liters of transfection reagent, so depending on which protocol is used the cost can be about 0.01% that of commercially-available transfection reagents.

PEI structure. Image source: https://en.wikipedia.org/wiki/Polyethylenimine

PEI structure. Source: Wikipedia.

PEI is a simple polymer with an amine group and two carbon spacer that is thought to bind to DNA to produce positively charged particles that can enter the cell.

Some of the negative aspects of PEI compared to more sophisticated transfection reagents are:

  1. PEI tends to be rather cytotoxic. While PolyJet™ DNA In Vitro Transfection Reagent  is composed of proprietary bio-degradable polymers designed to greatly reduce cytotoxicity, standard PEI has been shown to induce apoptosis in a variety of human cells.
  2. PEI is difficult to dissolve. Linear PEI is solid at room temperature and somewhat soluble in hot water and low pH. A typical protocol might involve dissolving 8mg of PEI in 25mL of water, a process that may take many hours or days and/or result in a non-uniform solution that cannot be sterile filtered.

To solve this problem, Polysciences has released the a much more soluble hydrochloride salt form (catalog nr 07924765-2), called Polyethylenimine “Max”, (Mw 40,000) – High Potency Linear PEI. This MW 40,000 form corresponds to the MW 25,000 polymer length in free base form, with the salt accounting for the molecular weight difference.

PEI max structure.

PEI max structure.

Looking through the literature one might reach the conclusion that PEI is a universal transfection reagent suitable for transfecting cultured cells or use in vivo.

Here’s a summary of some of the earlier scientific articles using PEI for various transfection applications:

Nucleic Acid Cell Type Publication
68-mers Hepatocytes ref
antisense oligos Neurons ref
BAC DNA mouse (in vivo) ref
DNA 293E ref
DNA Adult neural stem cells ref
DNA Brain derived cells ref
DNA chicken (in vivo) ref
DNA CHO cells ref
DNA Cos-1 ref
DNA Cos-7 (nuclei) ref
DNA Embryonic neurons (in vivo) ref
DNA Fetal mouse liver ref
DNA HeLa cells ref
DNA Human monocytes ref
DNA HUVEC ref
DNA L929 cells ref
DNA LNCaP cells ref
DNA Mouse brain ref
DNA Mouse lung ref
DNA Mouse lung ref
DNA Murine adult neural stem cells ref
DNA Ovarian carcinoma cells ref
DNA Postmitotic neurons ref
DNA Pseudocystic tumor cells ref
DNA Rat (in vivo) ref
DNA Rat brain ref
DNA Rat fetal hypothalamic cells ref
DNA Rat kidney ref
DNA Rat kidney ref
DNA WERI-Rb1 retinoblastoma ref
DNA Xenopus tadpole brain ref
dsRNA and siRNA Snail, Biomphalaria glabrata ref
modified siRNA Murine melanoma cells ref
siRNA in vivo ref
siRNA Pancreatic cancer cells ref
YAC HT1080 cells ref

 

 

 

 

 

 

 

 

 

In addition to use as a direct transfection reagent, PEI is used for a variety of “combination” gene delivery methods. Adenofection for example makes use of PEI to permit delivery of large plasmids/BAC/YAC into cells by non-covalently coupling of the DNA to an adenovirus. A variety of other approaches make use of modified (e.g. PEGylated) PEI for gene delivery or have targeted PEI to specific tissues with antibodies or proteins.

In the list of applications, mRNA delivery into cells seems conspicuously absent. Indeed at least one paper indicates that PEI-mediated transfection of mRNA is very inefficient. For mRNA transfection, researchers have better success with the Stemfect™ RNA Transfection Kit or the mRNA-In Transfection Reagent.

Transfection reagents by tebu-bio

HA tagged beta-tubulin cDNA was delivered into CHO cells. HA-beta-tubulin (Green) – Endogenous alpha-tubulin (Red).

 

Leave your comment below to share your experience with PEI!

 

 

 

 

 

 

 

 

 

 

 

 

 

CRISPR genome editing: which cell line to choose?

Many labs have adopted the CRISPR genome editing technology to make knock-out and knock-in cell lines.

This technology produces first a targeted break in genomic DNA, which can then be exploited to produce cell lines with genes knocked out or where a donor vector has been used to introduce new genetic elements (point mutants, fluorescent tags, antibiotic resistance cassettes, etc.). Essentially any desired modification to the cells genome can be made. In setting up these genome editing projects there are many choices to be made including vector for the Cas9 protein and for the sgRNAs. Perhaps the most difficult choice, however, can be which cell line to use. Even the most affordable stable genome editing cell line development services can come with a significant cost, so choosing the right cell line at the beginning is crucial. Here we explain some of the choices researchers have in setting up their CRISPR genome editing projects and give our advice for cell line selection.

[Read more…]

Kit-Free Site Directed Mutagenesis Protocol

Molecular biologists are familiar with the QuikChange® Site-Directed Mutagenesis Kit that allows rapid intoduction of a point mutant into a plasmid/vector/mammalian expression construct. Briefly, the protocol first involves a thermocycling/PCR step with mutagenic primers, followed by a DpnI digestion step to digest the methylated parental/wild-type plasmid, and finally transformation into competent cells for nick repair.

 An animation showing the basics of site directed mutagenesis. Image source: http://commons.wikimedia.org/wiki/File:Site-Directed-Mutagenesis.gif

An animation showing the basics of site directed mutagenesis. Image source: http://commons.wikimedia.org/wiki/File:Site-Directed-Mutagenesis.gif

 

Most experts we’ve talked to still use this technique, but don’t see the point of an expensive kit. Instead they use their own protocols with inexpensive enzymes and reagents bought separately. One such protocol involves the following:

“QuickChange” Protocol for Site-Directed Mutagenesis

Step 1: Design mutagenesis oligos (Primer 1 and Primer 2) using the website PrimerGenesis

Step 2: Order high quality custom oligos from tebu-bio

Step 3: Set up this reaction:

Plasmid DNA (50ng/ul) 1  ul
10x Reaction Buffer (comes with polymerase) 5 ul
50 mM MgCl2 (comes with polymerase) 1 ul
dNTPs (10mM) 1 ul
DMSO 2.5 ul
Primer 1 (10uM) 1 ul
Primer 2 (10uM) 1 ul
AccuStar™ DNA polymerase* 1 ul
Water 36.5 ul
TOTAL 50 ul

* Due to inherent 3’-to-5’ exonuclease activity of this very high fidelity enzyme, the polymerase must be added last to the reaction in order to prevent primer damage.

Step 4: Run the following PCR Cycle in a thermocycler

95°C 5 min; 15 cycles of (95°C 30sec, 60°C 1 min, 68°C 15 min*), 68°C 10 min, 10°C forever.

*for large (>9kb) plasmids, increase the extension time to 20min.

Note: run a negative control reaction without polymerase enzyme.

Step 5: Add 1 ul of Dpn1 enzyme to PCR tubes and leave at 37C for 2 hours

Step 6: Prepare a 1% agarose gel with GreenView™ Plus DNA Gel Stain

Step 7: Load 10ul of completed reaction mixed with SafeGreen™ Loading Dye next to 1kb DNA ladder and run the gel at 150V for 10 min to determine if PCR was successful. You should not see a product for the ‘negative control’ tube that had no polymerase. Troublshooting: If PCR reaction yields no product, then do gradient for the annealing temperature. The DMSO is extremely important, so keep this in the reactions.

Step 8: Mix PCR reaction with 500ul of PB buffer and purify on column. Wash with PE buffer and elute with 50 ul of water. Use 10ul of for transformation of TransforMax™ EC100™ competent cells.

 

Interested in using this protocol to your lab? Here is a list of products you will need:

Item Name Catalog Number
2′-dNTP Set 040N-2505-25 
Accustar DNA Polymerase 218ME-0067-05 
Molecular Biology Grade Agarose 218EP-0010-05 
GreenView Plus DNA Gel Stain 217N101 
SafeGreen Loading Dye 217D012 
TransforMax EC100 Chemically Competent E. coli 035CC02810 
1kb DNA Ladder 217D001

 

 

 

Legal Notes: QuikChange® is a registered trademark of Stratagene.

Using CRISPR to knockout an essential gene

Genome editing technology enabled by CRISPR and TALEN has become mainstream. Most cell biology labs are engaged in projects to create custom cell lines with knock-outs and knock-ins, and companies such as GeneCopoeia even propose complete cell line generation services. Projects can involve transfection of mammalian expression constructs, TALEN pairs, or direct transfection of RNA.

When scientists want to make a stable knockout cell line, one of the first questions they should ask is whether or not the resulting cell line will be viable. Often a murine knockout mouse has been made and/or siRNA knockdown experiments have been performed in human cells, so experienced users have a good idea if a human knockout cell line will be viable. There are certainly some cases, however, where either the researcher knows or expects that a complete knockout will not be viable but wishes to make the knockout nonetheless. What is the best way to deal with all of the risk involved with starting an relatively expensive and time-consuming project like this that could end in failure?

To knockout an essential (or potentially-essential) gene we recommend the following:

1) Plan the entire project in collaboration with a professional company offering stable cell line development services.

2) Develop your knockout strategy using TALEN or CRISPR.

3) Introduce a functional copy of your gene under the control of doxycycline into a safe harbor knock-in site. This copy of the gene should be designed to be resistant to the TALEN/CRISPR knockout strategy designed in #2.

4) Generate the parental doxycycline-inducible/repressible cell line and isolate a stable clone.

5) Knockout the functional endogenous alleles of the gene in this stable clone.

You will be then left with a viable cell line that will allow you to test whether or not complete knockout results in viable cells. It may be even possible to include loxP sites in the knocked-in cassette to completely remove the doxycycline controlled wild-type gene.

safe_-harbor

 

Any questions? Leave your comments below…

BioID proximity-dependent biotinylation studies

In 2012, Roux et al. published a nice paper, that received no less than four article recommendations from F1000 researchers. The paper described a method for tracking the interaction partners a protein has had within a cell (a history of its interacting partners). The method, called BioID, is based on proximity-dependent biotinylation of proteins by a promiscuous biotin ligase mutant BirA (R118G), which is fused to your protein of interest. After an overnight incubation with biotin, cells can be subjected to harsh lysis and biotinylated proteins can be isolated and identified by mass spectroscopy to determine the proteins that had come into contact with the chimeric BirA (R118G) protein. This method is a bit different from standard co-IP or pull-down experiments, because it allows one to identify proteins who interact transiently or weakly with the protein of interest. Also, due to the strong biotin-avidin binding affinity, harsh washes can greatly reduce background protein binding.

Image adapted from: DOI: 10.1371/journal.pone.0122886

Image adapted from:
DOI: 10.1371/journal.pone.0122886

 

We’ve consulted some experts using this protocol in their labs, and here are the steps required to perform BioID:

1) Obtain pcDNA3.1-myc-BirA(R118G) from an academic collaborator or Addgene

2) Perform or request a custom cloning of your gene of interest (GOI) into the vector EcoRI and HindIII sites to myc-BirA-GOI vector

3) Transfect cells with BirA(R118G) chimeric protein mammalian expression vector using an appropriate transfection reagent

4) Incubate cells overnight in medium with 50 mM biotin

5) Lyse cells directly in 1X Laemmli buffer and sonicate.

6) Incubate lysates with streptavidin-coupled beads for 3 hours at 4°C.

7) Pellet beads and wash 5 times with 1mL RIPA buffer.

8) Elute bound proteins by boiling at 100°C for 8 minutes in 2X Laemmli buffer.

9) Analyze eluted proteins by western blot of mass spec.

Now a number of research articles have been published using the BioID method and have identified novel protein-protein interactions. Some researchers have noted differing results using N- or C-terminal BirA (R118G) tagging. At least part of this might come from the chimeric protein’s intracellular localization. Indeed an important step is to confirm that the BirA(R118G) fusion has the same intracellular localization as the protein of interest.

 

 

 

 

New Fluorescent Labeling and Detection Products

For decades researchers have been using the famous Alexa Fluor® dyes developed by Molecular Probes for everything from flow cytometry to oligonucleotide labeling. These sulfonated forms of common fluorophores (coumarin, rhodamine, fluorescein, and cyanine) are thought to be more stable, brighter, and less pH-sensitive than the native molecules (Ref: Panchuk-Voloshina et al. 1999). Antibody users are likely most familiar with Alexa Fluor® 488 and Alexa Fluor® 594 which when used together allow for the simultaneous staining of one protein in green and another in red and can be used in combination with the blue nuclear dye DAPI.

Andy Fluor 488

The original Molecular Probes patents have likely limited the number of companies offering less expensive alternatives to the Alexa Fluor® dyes. Innovative dyes as DyLight® and IRDyes® have been developed and are used for specific applications, but direct alternatives for the standard Alexa Fluor® exitation and emission spectra dyes have been difficult/impossible to find…..until now.

Andy Fluor™ dyes are the next generation fluorescent dyes spanning the visible and near-infrared (IR) spectrum for labeling proteins and nucleic acids:

  • Andy Fluor™ 350
  • Andy Fluor™ 405
  • Andy Fluor™ 430
  • Andy Fluor™ 488
  • Andy Fluor™ 555
  • Andy Fluor™ 568
  • Andy Fluor™ 594
  • Andy Fluor™ 647
  • Andy Fluor™ 680
  • Andy Fluor™ 750

For oligonucleotide labeling by Nick translation, Random primer labeling, End-labeling with terminal deoxynucleotidyl transferase, Reverse transcription or PCR amplification Andy Fluor™ dNTPs are a nice low cost alternative to products such as ChromaTide® Alexa Fluor® 488-5-dUTP.

 

Label DNA RNA
Amine modification 5-Aminoallyl-dUTP 5-Aminoallyl-UTP
Biotin Biotin-11-dUTP Biotin-11-UTP
5-FAM 5-FAM-dUTP 5-FAM-UTP
5-FAM-X 5-FAM-X-dUTP 5-FAM-X-UTP
Andy Fluor 488 Andy Fluor™ 488-X-dUTP Andy Fluor™ 488-X-UTP
Andy Fluor 555 Andy Fluor™ 555-X-dUTP Andy Fluor™ 555-X-UTP
Andy Fluor 568 Andy Fluor™ 568-X-dUTP Andy Fluor™ 568-X-UTP
Andy Fluor 594 Andy Fluor™ 594-X-dUTP Andy Fluor™ 594-X-UTP
Andy Fluor 647 Andy Fluor™ 647-X-dUTP Andy Fluor™ 647-X-UTP
Cy3 Cy3-X-dUTP Cy3-X-UTP
Cy5 Cy5-X-dUTP Cy5-X-UTP

Originally developed by Applied BioProbes, the Andy Fluor™ dye labeled nucleotides are a small part of the rapidly growing fluorescence-based molecular biology offer from GeneCopoeia, Inc. Just as Alexa Fluor® has been the market leader for covalent fluorescent labeling of antibodies and oligonucleotides, SYBR® Green I, also produced by Molecular Probes, has historically been the leading dye for qPCR and “safe” agarose gel staining. GeneCopoeia has recently released their own: GreenView, GreenView Plus, and RedView agarose gel dyes.

 

Gel-stains

GreenView and RedView DNA Gel Stains very popular alternatives to Ethidium Bromide, as SYBR® Green-based Gel Stains have been demonstrated to offer a better safety profile by Ames test due to reduced DNA intercalation (Ref. Singer et al. 1998). Further optimization has resulted in dyes that also fail to cross the cell membrane of living cells resulting in an improved safety profile:

Cell permeability tests with Company A’s product, GreenView, GreenView Plus, and RedView. Hela cells were incubated at 37℃ with 1X Company A’s product, GreenView, GreenView Plus, and RedView for 30 min. GreenView Plus and RedView could not penetrate cell membranes to bind DNA in living cells, and were much safer than company A’s product, which rapidly entered cells and stained nuclei.

GreenView Plus and RedView fail to penetrate cell membranes to bind DNA in living cells.

 

Additional innovations from GeneCopoeia include the iQuant™ DNA Quantitation Kits to allow precise quantitation of DNA samples across a wide range of concentrations. Fluorescence-based DNA quantitation is highly sensitive and selective for DNA, relative to NanoDrop®-based absorbance readings (e.g. 260/280 ratios). Kits exist for double-stranded DNA or single-stranded DNA and can be adapted to microplate format using a fluorescence plate reader or tube format using fluorometers such as Qubit® or Quantus™:

 

iQuant

 

 

 

 Legal notes: Alexa Fluor®, SYBR®, Qubit® and ChromaTide® are registered trademarks of Life Technologies Corporation. DyLight® and NanoDrop® are registered trademarks of Thermo Fisher Scientific. IRDyes® is a registered trademark of Li-COR. Andy Fluor™ is a trademark of Applied Bioprobes. Quantus™ is a trademark of Promega.

Column-free, Caustic Solvent-free DNA or RNA Purification

Epicentre® (an Illumina Company) has developed a series of MasterPure™ DNA and RNA purification kits for nearly any sample type. While Epicentre’s QuickExtract™ DNA Extraction Solution is designed for the fastest extraction of low abundance DNA for PCR, the MasterPure™ kits yield the highest quality DNA and RNA for applications such as:

  • NGS genomic DNA and RNA-Seq library preps
  • DNA methylation/epigenomics studies
  • Genomic DNA and cDNA cloning
  • qPCR and qRT-PCR

These kits offer a simple protocol for the purification of Genomic DNA, cellular RNA, or total nucleic acid (TNA):

DNA RNA or Both

[Read more…]

PseudoU-RNA Molecular Weight Markers

Standard RNA ladders such as the DynaMarker® for large or small RNA or double-stranded size determination are routinely used by researchers wishing to confirm the molecular weight of an in vitro transcribed or chemically-synthesized RNA. Due to the altered gel mobility of pseudouridine-containing RNAs, a PseudoU-RNA Molecular Weight Marker can be used to more accurately assess the molecular weight of these modified RNAs.

PseudoURNALadder

[Read more…]

rRNA depletion, poly(A) enrichment, or exonuclease treatment?

RNASeq studies are hampered by the pervasive excess of reads mapping to ribosomal RNA (rRNA), notorious for greatly reducing the amount of useful mRNA sequencing data. Here we highlight the advantages and disadvantages of the various approaches have been developed to address this problem.

Source: DOI: 10.1371/journal.pone.0096094

Source: DOI: 10.1371/journal.pone.0096094

 

1) rRNA depletion with magnetic beads: Technologies such as Ribo-zero™, Ribominus™, and MICROBExpress™ seem to be based on similar technology that uses rRNA depletion probes in combination with magnetic beads to deplete rRNAs from a sample, thus achieving the goal of enriching the representation of mRNAs. Based on our interactions with end-users, Ribo-zero™ customers have been quite pleased with this technology, although some have found it a bit pricey. As a result, some scientists have adapted home-made protocols either using the Organism-Specific Probe Selection software or by designing probes empirically based on the reads they see in their samples. In both cases users custom order Biotin-TEG DNA oligonucleotides in combination with BioMag® Nuclease-Free Streptavidin Particles to complete their own protocols.

2) Poly(A) enrichment: Scientists working in eurkaryotes find that simply purifying poly(A) mRNA with BioMag® Oligo (dT)20 Particles is the most cost-effective approach. The BioMag® SelectaPure mRNA System is a complete low cost kit for isolating mRNA. Clearly users wishing to study histone mRNAs or those of prokaryotes will have to find another solution.

3) rRNA depletion by nuclease: Some of the most straightforward approaches for rRNA depletion involve simple digestion of rRNA with Terminator™ 5′-Phosphate Dependent Exonuclease. While a mature mRNA will carry an m7GTP cap on its 5′ end protecting it from the 5′-to-3′ exonuclease activity, rRNAs and tRNAs carrying only a 5′ phosphate will be degraded. A number of published reports have used this exonuclease approach which seems to be particularly interesting when users have complex environmental samples with multiple prokaryotic species. Alternative strategies involve the use of DNA probes complementary to the rRNA prior to digestion with RNAse H, which degrades only the RNA strand in an RNA:DNA hybrid. RNase-Free DNase I is then used to eliminate the DNA Probes. A report in Nature Methods (DOI:10.1038/nmeth.2483) comparing multiple approaches for RNASeq library prep determined that the Hybridase™ Thermostable RNase H-based approach was superior for low quality, degraded RNA samples.

4) aRNA amplification: Very common in microarray protocols such as those using Illumina BeadChip, the linear amplification of antisense RNA (aRNA) also called cRNA seems like an interesting approach. Kits like the TargetAmp™TargetAmp™-Nano, and TargetAmp™-Pico aRNA Amplification Kits are designed to amplify the input poly(A) mRNA from very low input (down to 10pg) RNA samples such as those obtained by laser capture microdissected tissue or by flow sorting. After amplification, sufficient sample is present for microarray analyses of gene expression. Based on technology developed by James Eberwine for single cell analysis, the TargetAmp kits have been used in RNASeq protocols with very low quantity samples (For example see: Matsumura et al. and Li et al.).

TargetAmp

5) Rolling Circle Amplification: Similar to the Eberwine-based methods, Rolling Circle amplification protocols involve first the reverse transcription of mRNA with the goal of amplifying the cDNA, primarily for single-cell applications (see Pan et al.). These protocols are quickly gaining popularity and make use of CircLigase to circularize full-length single-stranded cDNA or T4 DNA Ligase kits such as Fast-Link™ DNA Ligation Kits for double-stranded cDNA circularization. Use of Phi29 DNA polymerase is then possible, despite the fact that Phi29 normally requires templates of at least 3-4kb. While it is clear to see how reverse transcription using oligo(dT) will enrich mRNA reads relative to those mapping to tRNA and rRNA, such an enrichment would not be possible in prokaryotes. The rolling circle amplification protocol has been adapted to transcriptome analysis in single prokaryotic cells by making use of random hexamers combined with Terminator™ 5′-Phosphate Dependent Exonuclease described in #3 above (see Kang et al.). Interestingly, the protocol makes use of thiophosphate-linked RNA random hexamers to reduce the formation of primer-dimers and to protect against Phi29 DNA polymerase exonuclease activity.

Diagram illustrating rolling circle DNA replication (credit: credit Madeleine Price Ball)

Diagram illustrating rolling circle DNA replication (credit: Madeleine Price Ball)

Need help building your protocol for enrichment of mRNA reads? Contact the technical specialists at tebu-bio!

Legal notes: TargetAmp™ and Ribo-zero™ are trademarks of Epicentre-Illumina. Ribominus™ and MicrobeExpress™ are trademarks of Life technologies.