Plasmids 101: use of transposons in the laboratory (2023)

We often think of DNA as inert. It generally remains quiescent, making it easy to locate in a genome. But there's a kind of mobile DNA called a transposon that's a little hyperactive and likes to jump from place to place in the genome. This jump caught the attention of Barbara McClinntock, who first discovered transposons due to color changes in corn kernels. Besides maize, transposons are found in the genomes of many other prokaryotes and eukaryotes, which make up about 45% of the human genome (Muñoz-Lopez et al., 2010).

So why are transposons important? The outcome really depends on where a transposon lands. For example, in bacteria, transposons can move genes for antibiotic resistance between plasmids or from plasmids into the bacterial genome. If a transposon jumps to a gene in humans, it can be mutagenic and lead to human disease. While transposon insertions can be harmful, they also drive genomic evolution and are used by scientists as a tool to move DNA around in the lab.

Let's look at the main components of transposons, how they are classified and how they are used in the laboratory.

What is a Transposon?

DNA transposons are mobile, repetitive genetic elements found in both prokaryotic and eukaryotic genomes.

Parts of a transposon system

  • the transpose:The transposon is the DNA sequence that moves. This DNA sequence codes for the proteins that the transposon needs for transposition.
  • rearrangement protein: This is the protein needed to move the transposon sequence from one place in the DNA to another. This protein is either a transposase or a reverse transcriptase, depending on the transposition mechanism.
  • destination place: Different transposons are inserted into different DNA sequences or target sites. Integration of most transposable elements (TEs) results in the duplication of this target site sequence at the insertion site.

types of transpos

Transposable elements are divided into two classes based on their mechanism of transposition: class I TEs, also called retrotransposons, and class II TEs, also called DNA transposons. DNA transposons are often used as tools in the lab, so we'll focus on DNA transposons for much of the remainder of this article.

Class I transposable elements (TEs): retrotransposons

Class I TEs are also known as retrotransposons. They are converted using a "copy-and-paste" mechanism (Fig. 1). They first copy themselves as RNA transcripts and then rely on a reverse transcriptase enzyme to transcribe them back into DNA before inserting them into new target sites. This is similar to the way retroviruses, such as HIV, multiply. Class I TEs do not encode a transposase enzyme.

Class I TEs are considered replicative, as they make a copy of themselves each time they jump. This increases the copy number of the TE and at the same time increases the size of the host's genome.

There are two types of Class 1 TEs: those with long terminal repeats (LTR) and those without (TE non-LTR). LTR retrotransposons are something likeretrovirusboth in structure and replication mechanism. They contain two genes:to bitejpol. HepolThe polyprotein encodes the enzymes reverse transcriptase and integrase required for transposition of the LTR retrotransposon. Non-LTR retrotransposons contain two open reading frames (ORFs) that often end in a poly(A). ORF2 encodes endonuclease and reverse transcriptase activities.

Plasmids 101: use of transposons in the laboratory (1)

Figure 1: Overview of retrotransposon conversion.Retrotransposons are mobilized by a "copy and paste" mechanism. They first copy themselves as RNA transcripts and then use their reverse transcriptase (RT) enzyme to convert them back into DNA. They can then be inserted into new target sites. Created on

Classes II TE: DNA transposons

Class II TEs are also referred to as DNA transposons because they do not use an RNA intermediate when moving. Most class II transposons have a non-replicative transposition mechanism: they are first removed from one location and then inserted into another location (Figure 2). Class II TEs have LTRs on both ends.

Plasmids 101: use of transposons in the laboratory (2)
Figure 2: Overview of DNA transposon shuffling.Modified from Sandoval-Villegas et al., 2021. To mobilize a transposon, the enzyme transposase first ligates the long terminal repeats (LTRs) of the transposon, induces a double-strand break, and cleaves the transposon from the donor DNA. A DNA fingerprint remains. When the transposon-transposase complex finds its target site, it integrates, resulting in a target site duplication (TSD). Created on

Autonomous versus non-autonomous TEs

Autonomous transposons can be thought of as "full-length transposons" because they code for the protein they are supposed to move, a transposase or a reverse transcriptase. However, non-autonomous transposons require another TE to express a transposase or reverse transcriptase in order to transpose. Both class I and II transposons can be autonomous or non-autonomous.

DNA transposons commonly used in the laboratory.

While there are many different types of transposons, DNA transposons are most often used in the lab for genome manipulation. When transposons are used in the laboratory, the transposase gene is vertso that a gene of interest can be inserted between the LTRs of the transposon similar to whenViral packaging vectors.

Let's take a closer look at three different transposon systems adapted for use as research tools:Sleeping Beauty,PiggyBac, jTol2.

Sleeping Beauty

Sleeping Beautyis a synthetically transposable element developed from Tc1/knockout.sailortransposons found in fish (Sandoval-Villegas et al., 2021).Sleeping BeautyThe preferred target site for integration is TA dinucleotides and leaves the CAG DNA fingerprint of its terminal sequences at the cleavage site after cleavage by transposase. It has a payload of >100 kB, although the integration efficiency decreases as the payload size increases.Sleeping Beautyit has a virtually random integration profile in mammalian genomes. SB is active in vertebrates and integrates into human cells at a rate similar to that of retroviral vectors. The overactive version of SB transposase,SB100X, is ~100 times more efficient compared to the first generation SB transposase (Mates et al., 2009).hySB100ximproves over SB100X by having 30% more transposition activity (Voigt and others, 2016).

  • Sleeping Beauty Plasmids


Although the name suggests otherwise,varkentjeBacwas discovered in the diamondback moth (Potter and others, 1976). Its target site is TTAA and, unlike other transposons, it does not leave a DNA fingerprint sequence after cleavage.varkentjeBacIt can mobilize DNA larger than 100 kB and is vitrolikelivein yeast, plant, insect and mammalian cells, including human cells.varkentjeBacit is biased to integrate into transcription start sites, CpG islands and DNaseI hypersensitivity sites. IfSleeping Beauty,varkentjeBacit has an integration efficiency in human cells comparable to that of retroviral vectors. Heoveractive PB transposasa (hyPB)is ~10 times more active in mammalian cells compared to codon-optimized wild-typevarkentjeBactransposasa.

  • piggyBac-plasmiden


Tol2it was the first active DNA transposon reported in vertebrates. It was discovered in the Japanese Medaka fish because its insertion into the fish's tyrosinase gene caused albinism (Koga et al., 1996). unlikeSleeping BeautyjvarkentjeBac,Tol2it has a weak consensus sequence for its preferred target integration site: TNA(C/G)TTATAA(G/C)TNA. Tol2 can deliver 10-11 kB to mammalian cells without decreasing efficiency, with a maximum loading capacity of ~200 kB DNA. Equivalent tovarkentjeBac,Tol2it also prefers to integrate into transcription start sites, CpG islands and DNaseI hypersensitivity sites. Tol2 is only active in vertebrates and has a lower integration efficiency in human cells thanvarkentjeBacjSleeping Beauty. MinimumTol2OminiTol2is a shortened version of the originalTol2and has a ~3-fold increase in transposition activity

  • Tol2-transposonplasmiden

transposon applications

Now that you're familiar with some of the more popular transposon systems, let's see how they can be used in the lab.


Transposons are mutagenic elements in nature, making them a great tool for mutagenesis tests that detect gain or loss of function mutations. In these screens, transposons encode reporter genes, mutagenic cassettes, or barcodes. When transposons are delivered to cells or a model organism, they are inserted into the genome of the host. Transposon insertion sites are then detected with next-generation sequencing and analyzed to identify which inserts were positively or negatively selected during the experiment (Sandoval-Villegas et al., 2021). It is important to consider target site and host range preference when selecting a transposon for a mutagenesis test. For example,varkentjeBacjTol2they are better at detecting promoters and enhancers because they are amenable to insertion at these sites.

transgenic animals

transgenic animalsthey are often generated by injecting DNA directly into the pronuclei of fertilized eggs, leading to the random insertion of that sequence into the genome of the zygote, a process that is highly unpredictable. However, transposons are efficiently incorporated into the genome of the zygote after injection into the cytoplasm of a fertilized egg, a process that is inefficient when DNA is injected.Sleeping Beauty,varkentjeBac, jTol2All have been used to generate transgenic animals, including zebrafish, mice, rats and rabbits (Sandoval-Villegas et al., 2021).

transfer of genes

Transposons are an alternative to the delivery of transgenes by viral vectors, such asiPSC reprogrammingand gene and cell therapy, and have the potential to overcome some of the limitations of viruses. TEs have a large payload, up to 100 kBSleeping BeautyjvarkentjeBac, which is a huge advantage over viral vectors (~5 kB payload for AAV and ~8 kB payload for lentivirus). Large payloads, such as the ~11 kB cDNA for dystrophin, the gene mutated in muscular dystrophy, require truncation to fit viral vectors, but will easily fit into a TE. Transposons are also less likely to elicit an immune response than viral vectors, and they are also easier and cheaper to generate. Genetic alteration due to integration can occur with either method of administration, but since TEs are mainly introduced into intergenic regions, genetic alteration is less of a concern.

RNA-guided insertion of transposons.

A disadvantage of DNA transposons is that they do have a specific integration target site, such as TTAASleeping Beauty, can be inserted into the genome at any number of these sites.

To direct transposons to a single location in bacterial genomes, the Sternberg lab developed theINTEGRATE(Insertion of transposable elements by RNA-assisted guidewire). INTEGRATE is based on a natural Tn7 transposon found inVibrio choleraeencoding a CRISPR-Cas type I-F system. This system consists of four main components: 1) a CRISPR RNA (crRNA), 2) four proteins (TniQ, Cas8, Cas7, Cas6) that together with the crRNA form the QCascade DNA targeting module, 3) a transposase complex consisting of three transposase proteins (TnsA, TnsB, TnsC) and 4) the donor or minitransposon DNA containing the DNA payload of interest flanked by ~50-150 bp transposon end sequences (Fig. 3A).

To transpose the INTEGRATE system, the QCascade complex first explores the genome and binds to the target DNA sequence. The transposase complex binds to the minitransposon, detaches it from the donor molecule and inserts it approximately 50 bp downstream of the QCascade genomic target site (Fig. 3B). The INTEGRATE system can achieve ~100% integration of DNA up to 10 kB in bacteria.

Plasmids 101: use of transposons in the laboratory (3)
Figure 3: INTEGRATED system for RNA-guided transposon insertion in bacterial genomes.A) INTEGRATE system components. B) Mechanisms of RNA-guided shuffling with INTEGRATE. The QCascade complex probes the genome and binds to the target DNA sequence. The transposase complex binds to the minitransposon, detaches it from the donor molecule and inserts it approximately 50 bp downstream of the QCascade genomic target site. T-RL= right-left orientation of the transposon. T-LR= left-right orientation of the transposons. Picture ofstem stem.


Transposons are another tool for moving DNA around or within eukaryotic and prokaryotic genomes. They only need two key components, the DNA sequence of the transposon and the transposon protein, to move the DNA. There are three popular systems for laboratory use (Sleeping Beauty,varkentjeBac, jTol2), each of which has different applications (detection of mutagenesis, transgenic animals, gene transfer). The discovery of RNA-guided transposons in bacteria lays the groundwork for combining the superpowers of CRISPR and transposons to enable targeted delivery of large amounts of DNA (>100 kB) to specific locations in the genome.

Plasmids 101: use of transposons in the laboratory (4)

References and Resources


Feschotte C, Pritham EJ (2007) DNA transposons and the evolution of eukaryotic genomes. Annu Rev. Genet 41:331–368.

Kumar A (2020) Skip: Transposons in and out of the lab. F1000Res 9:135.

Muñoz-López M, García-Pérez J (2010) DNA-transposons: ground in applications in genomics. GC 11:115–128.

Pray, L. (2008) Transposons: the jumping genes. Nature Education 1(1):204.

Sandoval-Villegas N, Nurieva W, Amberger M, Ivics Z (2021) Contemporary transposon tools : an overview and guide through transposon mechanisms and applications.Sleeping Beauty,varkentjeBacjTol2for Genome Engineering. IJMS 22:5084.

Additional resources on the Addgene blog

  • Learn more about theINTEGRATED bacterial genome engineering system
  • lees over hemSleeping Beauty DNA transposon
  • Learn more about thepiggyBac DNA-transposon

Subjects:Plasmids 101

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