Imaging Fluorescent Proteins in vivo expressed from DNA vaccine constructs E. Kinnear, R. Cole, A. Walters, R. Shattock, J. TregoningMucosal Immunity Group, Department of Medicine, St Mary’s Campus, Imperial College London, London, W2 1PG, UK.

DNA vaccines have huge potential in clinical applications since they are cheap, easy to manufacture, and in animal studies have proved immunogenic and safe. However, clinical trials in the past have so far been disappointing. The optimisation of DNA delivery and expression may improve the immunogenicity in clinical trials. To improve DNA delivery it is necessary to improve the technology used to monitor its expression in vivo. One way of doing this is to use fluorescent proteins[1]. Green fluorescent

protein is the most widely used, however it is ineffective in vivo due to absorbance characteristics of the skin. Far red fluorescent proteins could be used to circumvent this problem, allowing better imaging[2]. However, many of the studies reporting the use of these proteins have either injected cells expressing the proteins[3], used recombinant virus[4] or recombinant protein in an artificial mouse[5]. We were interested in optimising the conditions for visualising protein after DNA vaccination.

Material and Methods:

A plasmid expressing the red fluorescent protein tdTomato

under a CMV promoter was obtained from Addgene (plasmid

30530 - pCSCMV:tdTomato)[6]. It was prepared for in vivo use

with an EndoFree Plasmid Giga prep kit (Qiagen).

Female BALB/c mice were obtained from Harlan Scientific

and used at 6-8 weeks of age. All procedures undertaken

had been approved by the local ethics review board and per-

formed by personal licensees under the appropriate project

license. Experiments carried out were in accordance with

Animals (Scientific Procedures) Act 1986. Prior to immuniza-

tion mice were shaved and treated with depilatory cream in

the area of interest.

50 μg DNA was delivered into mice either intramuscularly

(I.M.) or subcutaneously (S.C.), with or without electropora-

tion, which has been shown to increase the expression of

DNA vaccines[7]. Where used electroporation was performed

with two lots of 5 pulses of 150 V with switched polarity

between pulses were delivered using a CUY21 EDIT system

(BEX, Japan). Electroporation was delivered using a fixed gap

needle electrode (LF560S5) inserted into the muscle after

the injection.

Mice were imaged at excitation 550 nm and emission

600 nm using the Bruker In-Vivo MS FX PRO system (Bruker

BioSpin, MA, USA). Images were obtained with 1x1 binning,

f-stop 2.8 and 60 second acquisition time. To avoid autofluo-

rescence at the imaging site, mice were initially shaved and

depilated (Immac Cream, Veet). Image analysis was carried

out using the Bruker Molecular Imaging Software Suite.

Results and Discussion

Initially we wished to determine the effect of electropora-

tion on signal intensity after immunisation. We discovered

a number of sources of noise that can detract from signal,

these include the animal faeces and urine, the fur and

the indelible marker pen we use to differentiate animals

(Figure 1). We used a number of approaches to reduce this

background noise, including shaving/ depilating the mouse

and the use of ear punch to identify mice.

Having reduced the noise signal we then compared expres-

sion of tdTomato from plasmid DNA delivered with and with-

out electroporation. It has been shown that electroporation

can significantly enhance the expression of DNA vaccines

and we wished to determine whether it could affect expres-

sion of fluorescent proteins in vivo.

Figure 1. Electroporation (EP) significantly enhances intensity of fluorescent protein expres-

sion in vivo. (A) Mice injected subcutaneously on their backs with 50 μg tdTomato with or

without electroporation (EP) and imaged over 72 hours (B) Mice injected subcutaneously in

the central epigastric region with 50 or 100 μg tdTomato with or without electroporation. Red

circle indicates the region of fluorescent protein expression.

Electroporation significantly enhanced the signal observed

and there appeared to be no difference in expression

between the 50 and 100 μg groups. So for future studies we

used 50 μg with electroporation.

The other aspect of DNA vaccination we were interested in

was the route of immunisation. We compared subcutaneous

immunisation with intramuscular immunisation and observed

that when successful, intramuscular immunisation gave a

stronger signal, but was less reliable overall than subcutane-

ous immunisation (Figure 2).

Conclusion

We showed that tdTomato-encoding DNA could be efficiently

delivered via electroporation, and thus provide a tool for in

vivo vaccination with DNA. However, the use of red fluores-

cent proteins for in vivo imaging of delivered DNA is chal-

lenging. There are a number of sources of auto-fluorescent

noise that can occur within the experiment even within the

red spectrum. Careful preparation of the animals and strongly

expressing/fluorescing proteins can reduce this problem, but

not completely mitigate it. The recent availability of further

red emitting fluorescent proteins like the IFP or mKate2 might

further improve in vivo imaging capabilities.

Figure 2. Comparison of routes of immunisation. 5 BALB/c mice were intramuscularly (I.M.) (A)

and subcutaneously (S.C.) (B) immunised with electroporation with 50 μg of tdTomato plasmid.

Imaging was carried out at 48 hours and 72 hours at excitation= 550 nm and emission= 600 nm.

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References

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