Process Of DNA Delivery To Eukaryotic Cells Biology Essay

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The delivery of genes into human cells has many potential therapeutic benefits and, therefore, has prompted the search for non-viral DNA delivery vehicles which posses the desirable properties of viruses, such as their ability to bypass cellular barriers, but pose fewer potential health risks to the host. This has led to the discovery of polycationic compounds such as polyethylenimine, which are safer, more efficient DNA delivery vehicles, condensing DNA into small particles to cross the cell membrane and providing defence against many of the barriers to gene delivery by endosomal escape, for example. H58 has also been implicated in gene therapy, due to its ability to upregulate gene expression at sub-lethal concentrations, it may be the answer to increased transgene expression in eukaryotic cells. Recruitment of transcription factors and TBP/TATA box element complex formation may play a key role, however the precise mechanism has not yet been fully elucidated. Polyethylenimine, H58 and heparin were investigated using transient transfection of Raw264.7 cells (murine macrophage line) with pGL3-control and pGL3-basic (with an Nramp1 promoter) vectors. Polyethylenimine increased luciferase activity by up to 20-fold. The effect of H58, when added at 0 hours, was a 30-fold increase in luciferase activity. Heparin caused up to 99% inhibition of luciferase activity by preventing uptake of PEI-DNA complexes into the cells. When added at later intervals, heparin had a lesser effect as the majority of DNA delivery had already occurred; suggesting the action of heparin is almost exclusively extracellular. An increase in luciferase activity when heparin was added after 4 hours prompts the need for further investigation into which pathways may be activated to exhibit this effect.

Abstract 2

List of abbreviations 3

Introduction 3

Aim 7

Method 8

Results 10

1. The effect of PEI (delivery agent) on transfection efficiency 10

2. The effects of H58 on transfection efficiency 13

3. The effects of Heparin on transfection efficiency when added after 0hrs 16

4. The effects of Heparin (added at 0, 2 and 4 hours during the transfection procedure) on transfection efficiency 18

Discussion 22

Conclusion 25

References 26

Appendix 27

List of abbreviations

H58 H58

HSPG Heparan sulfate-bearing proteoglycans

MGBL Minor groove binding ligand

NPC Nuclear pore complex

PEI Polyethylenimine

Introduction

The ability to deliver DNA into the nucleus of eukaryotic cells comes naturally for viruses, making them the perfect vehicle through which to carry out this process in applications such as gene therapy. The use of viral vectors, however, comes with a cost when dealing with the delivery of therapeutic genes into human cells, as viruses can cause pathogenicity, oncogenicity and stimulation of an immunological response in the host1.

Activation of oncogenes can be caused by incorporation of the viral genome alongside an oncogene, giving the viral promoter control over the expression of that gene, possibly leading to tumour formation in the host. In recent years, a series of non-viral vectors have been researched in an attempt to mimic the intelligent pathways of viral vectors without the potential health risks1.

There are some properties of viral vectors which are desirable in their non-viral counterparts, such as transfection efficiency, which many non-viral vectors seem to lack. Cationic lipids (such as Lipofectamine) are less adapted than viruses to overcoming intracellular barriers in place to prevent DNA entering the nucleus and, therefore, transfection efficiency can be greatly reduced to the extent that only 1% of DNA may be delivered to the host cell nucleus2,3.

There are many desirable properties for a DNA transport vehicle, but no single vector possessing all desirable properties without the health risks associated with viral vectors has been synthesised or discovered5. Lipoplexes (liposome-DNA complexes) form through interactions between the lipid molecules, meaning that there is very limited control over the properties of the lipoplex once it's formed and that adjuvants are often required to increase efficiency of transfection using these vectors5. In the case of polycations (such as PEI), they are self-forming, meaning that there is greater scope for chemical manipulation of their macroscopic properties, giving this type of vector greater transfection efficiency and targeting than lipoplexes and with fewer cytotoxicity risks than viral vectors4.

Cytotoxicity is a source of major concern when investigating potential vectors for gene delivery and therapy. In the case of PEI, it has been found that the level of damage to the plasma membrane caused by PEI derivatives increases proportionally with molecular weight (PEI800 causes more membrane damage than PEI25, for example), highlighting the need to compromise between transfection efficiency and damage to the cell5. This research has led to the discovery of polycationic compounds as vehicles for the transport of DNA into eukaryotic cells, such as the promising DNA delivery agent polyethylenimine (PEI), shown in figure 1 below4.

Figure 1 - The chemical structure of polyethylenimine - adapted from Thomas and Klibanov4.

Figure 2 illustrates the barriers that a vector has to overcome in order to deliver DNA into the nucleus of the host cell. These include The initial barrier to DNA delivery into cells is that of cellular uptake3,5. Upon binding polycations, such as PEI, DNA forms a polyplex which enters the cell via endocytosis and is encapsulated within an endosome (small, membrane-bound vesicle) which would usually prevent the DNA from being transported into the nucleus3. However, in the case of PEI, the endosome is destabilised and the DNA-PEI polyplex can escape (Fig 2, 3a), allowing it to be reach the nucleus where the DNA can decondense and separate from the PEI vehicle. Once inside the nuclear membrane, the DNA can be transcribed and, ultimately, translated into protein3,5. It is by this mechanism that PEI has proven to be such an effective non-viral DNA delivery vector; overcoming the barrier which has rendered many other vectors useless in this process5. The mechanism by which PEI destabilises the endosome, allowing the polyplex to escape the low pH and hydrolytic enzymes within, is explained by the 'proton sponge effect'5. This suggests that the nitrogens within PEI are protonated whilst it's inside the endosome/lysosome, causing an influx of chloride ions, altering its osmotic potential and causing it to rupture, releasing the contained polyplex (polycation-DNA complex) into the cytoplasm of the cell. PEI provides protection of the DNA from degradation by nucleases in the cytosol.5

Figure 2 - Barriers to DNA delivery - adapted from Wagstaff and Jans2.

It has since been discovered that the formation of polyplexes not only condenses DNA into extremely small particles (rods, toroids and spheroids - 20-200nm in diameter5) but also conveys a strong positive charge onto it, causing polyplexes to interact with the negatively charged proteoglycans on the cell surface, such as the heparan sulphate-bearing proteoglycans (HSPG)5,6,7. It is by this mechanism that it is believed that heparin (a polyanion) competes with DNA for binding with PEI (a polycation) and possibly causing the proteoglycans on the cell surface to be cross-linked, rendering them unable to bind polyplexes and explaining the reduction in transfection efficiency caused by the presence of heparin6,7.

The DNA minor groove binding ligands known as H42 and H58 were primarily used as fluorescent dyes for staining DNA, due to their specificity for binding AT-rich sequencing in the minor groove of B-DNA8,9,10. More recently, however, they have been found to have an effect on transfection efficiency (DNA delivery) and luciferase gene expression in cells transfected with plasmid DNA which has been incubated with either Hoechst ligand prior to the transfection process9. These findings have implications for the use of such DNA minor groove binding ligands in gene therapy; for use in delivery DNA into the host cells more efficiently, with greater chance of expression of the gene of interest within the host9,10. These ligands bind reversibly within the minor groove of double-stranded helical DNA via their benzimidazole region, specifically to AT-rich sequences to form non-covalent complexes which can be more efficiently delivered into the host cell. As seen with polyethylenimine, above, it is usually required for a non-viral DNA delivery agent to bind cell surface receptors, such as proteoglycans, for effective uptake of the polyplex into the cell10. However, in the case of H58, this requirement for receptor-mediated uptake is bypassed, allowing it to pass freely into the cell and across the nuclear envelope, making it extremely efficient at entering the nucleus, even in non-dividing cells in which the NPC is completely intact10. This property of Hoechst 22358 is ideal for delivery of DNA across the protective barriers of the cell in order to achieve efficient delivery into the nucleus, but it was unclear as to whether Hoechst could convey its membrane-permeant property onto DNA by complex formation. It has been found that Hoechst-DNA complex formation causes DNA to acquire this property; reduced polarity and, therefore, increased hydrophobicity, allowing it to permeate membranes more effectively, increasing efficiency of DNA delivery into the nucleus9,10.

Figure 3 - The binding of H58 to the DNA minor groove - adapted from Dervan11.

Aim

The aim of this investigation is to determine the effects of PEI, heparin and H58 on the efficiency of transfection of pGL3-contol and pGL3-basic (with Nramp1 promoter) vectors into Raw264.7 cells. This can be measured using a luciferase reported assay to deduce the level of transcription occurring within the cell nuclei as light energy is released when luciferase enzyme is produced.

Method

Materials

Plastics/glass:

01. White, yellow and blue pipette tips (2 boxes of each)

02. 1.5ml and 2ml eppendorf tubes

03. Gilsen pipettes (10µl, 20µl, 200µl, 1000µl)

04. 20ml universal tubes

05. 3ml disposable plastic graduated pastettes

06. Sterile cell plate spreaders

Electrical/mechanical

07. Microcentrifuge

08. Laminar Flow Hood

09. Haemocytometer

10. Luminometer

11. Microscope

12. Microtitre plate reader

13. Promega luciferase assay system

Solutions:

14. BSA standard (0.4mg/ml)

15. Trypan blue solution (sigma 95039)

16. Bio-Rad protein assay reagent

17. Hibitane solution

Growth of competent DH5α cells and attainment of plasmid DNA procedure

The plasmid DNA used in this procedure (pGL3-control and pGL3-basic with an Nramp1 promoter) was obtained using competent DH5α cells (E.coli strain), cultured overnight at 37°C in a shaking incubator as set out in: Sambrook, J and Russell, D. (2001) Molecular biology: A laboratory manual (third edition).

(Also available at: http://www.cshlpress.com/default.tpl?cart=126219297452061 432&fromlink=T&linkaction=full&linksortby=oop_title&--eqSKUdatarq=21)

Transfection procedure

The eukaryotic cells used for the transfection experiments are Raw264.7 from murine macrophage line (in Balb/c mice). These mammalian cells grow much slower than bacterial cells (such as the DH5α cells used in the attainment of plasmid DNA), so are grown in nunclon flasks containing DMEM growth media and 20% FCS (foetal calf serum) in a sterile incubator with an atmosphere of 5% CO2.

Ensuring gloves are cleaned with 70% ethanol and kept inside the laminar flow hood, cells should be removed from the flask using a single-use sterile cell scraper and poured from the nunclon flask into a 50ml falcon tube.

A haemocytometer may be used to determine the concentration of viable cells in the DMEM decanted from the nunclon flask with viability distinguished by addition of trypan blue which not taken up into viable cells, leaving the non-viable cells stained blue under the inverted microscope. The details of how to use a haemocytometer can be seen at: http://www.sigmaaldrich.com/sigma/datasheet/Z35 9629dat.pdf (also at: http://sigmaaldrich.dirxion.com/WebProject.a sp?BookC ode=cel08pfp#). A 1:1 trypan blue and cell suspension mix (100μl+100μl) was made and cell count determined.

The cells were grown overnight, prior to the transfection, on a 12-well plate in a sterile, humid incubator at 37°C in an atmosphere of 5% O2. Upon removal from the incubator, the DMEM and 20% FCS media were removed from the wells using a suction pump. To wash the cells, 1ml of DMEM media (no FCS) was added to each well and removed twice followed by the addition of 0.5ml of DMEM. Prepared tubes (one per set of 3 replicates), one tube (B) containing 100μl DMEM and 3μl PEI added to another tube (A) containing 100μl DMEM and 1μg plasmid DNA (either pGL3-control or pGL3-basic with Nramp1 promoter). The tubes containing DNA-transfection agent complex (polyplexes) were left to incubate for 20mins before 60μl of each tube (B+A) was added to each set of three replicate wells (20μl excess solution to allow for pipetting inaccuracies) and the plate was incubated at 37°C in 5% CO2 for 4 hours.

After this incubation period, 0.5ml of DMEM+20% FCS was added to each of the 12 wells to provide growth factors and the plate was returned to the incubator for 24hours in the conditions stated previously. After 24hours incubation, the plate was removed from the incubator, the medium removed using a suction pump (as above) and the cells rinsed with 0.5ml PBS twice, blotting excess PBS onto laboratory paper.

The cells were then lysed using a 5-fold dilution of 5xconcentration extraction buffer (800ml deionised water + 200ml extraction buffer) and harvested using a clean (not sterile) plastic cell scraper for each set of 3 replicate wells. The extract was then transferred into a 1.5ml eppendorf tube and stored on ice. The tubes of extract were then spun for 3 minutes in a microcentrifuge to collect all unwanted cell debris in the apex of the tube (forming a pellet) and the supernatant broth was pipetted off into fresh eppendorf tubes and stored on ice.

Luciferase reporter assay procedure

The luciferase activity within the transfected cells was measured using a luminometer (as seen at: http://www.promega.com/tbs/tm 033/tm033.pdf) and 50µl of luciferase assay reagent rather than 100µl.

Bio-Rad assay procedure

The protocol for the Bio-Rad assay used (as found at: http://cores.montana.edu/uploads/Proteomics%20Core/BioradBradford.pdf) was carried out, but with 250µl of Bio-Rad assay reagent with 2.5µl of sample DNA and 0-20µl of BSA standard solution in wells 1-12 in increments of 3µl.

Results

1. The effect of PEI (delivery agent) on transfection efficiency

Before the effect of H28 or heparin could be investigated, it was necessary to ensure that the transfection process was successful and establish the difference in luciferase activity that was to occur by simply adding transfection reagent (PEI) to give an idea of the level of luciferase activity to expect without the use of heparin or H28.

For this experiment, cells were cultured overnight in DMEM growth media with pGL3-control plasmid DNA in the absence of PEI and in the presence of PEI and also in DMEM growth media with pGL3-basic with Nramp1 promoter plasmid DNA in the absence and presence of PEI. The luciferase activity in cells from each condition was established using luciferase reagent and a luminometer, as in the procedure above. The results of this experiment are shown in figure 4a and 4b below.

Figure 4a: The luciferase activity values for each sample condition (normalised to protein amount, LU/µg) are shown and the result of a t-test statistical analysis for each condition

Effects of PEI (DNA delivery agent) on transfection

Condition

LU/5µL

LU/µL

LU/µg

Mean

Std dev

T-test (p value)

pGL3, no PEI 

2.377

0.475

0.792

 

0.981

 

 

0.180183959

 

 

 

 

pGL3, no PEI vs.

pGL3 + PEI =

0.143335856

2.997

0.599

0.999

3.454

0.691

1.151

pGL3 + PEI

116.2

23.240

46.480

 

30.517

 

 

21.620879

 

97.9

19.580

39.160

14.78

2.956

5.912

Nramp1, no PEI

14.31

2.862

3.578

 

4.100

 

 

1.647124388

 

 

 

 

Nramp1, no PEI vs.

Nramp1 + PEI =

0.178682851

23.78

4.756

5.945

11.11

2.222

2.778

Nramp1 + PEI 

53.35

10.670

13.338

9.537

 

5.414241

 

47.74

9.548

11.935

13.35

2.670

3.338

The t-test carried out on this data did not indicate any significance, so those of another group (Emily Wilkinson and Sam Wilkinson, see: appendix) were used in order to carry out the analysis, below.

Figure 4b: The luciferase activity values for each sample condition (normalised to protein amount, LU/µg) are shown and the result of a t-test statistical analysis for each condition, obtained by E. and S. Wilkinson.

Effects of PEI (DNA delivery agent) on transfection

Condition

LU/5µL

LU/µL

LU/µg

Mean

Std dev

T-test (p value)

pGL3, no PEI 

2.377

0.475

0.9508

 

1.177

 

 

0.216220751

 

 

 

  pGL3, no PEI vs.

pGL3 + PEI =

 

0.050419572

2.997

0.599

1.1988

3.454

0.691

1.3816

pGL3 + PEI

102

20.400

15.692

 

24.338

 

 

9.575944

 

147.5

29.500

22.692

225.1

45.020

34.631

Nramp1, no PEI 

2.467

0.493

0.705

 

0.946

 

 

0.495812753

 

 

 

  Nramp1, no PEI vs.

Nramp1 + PEI =

0.238892538

2.161

0.432

0.617

5.308

1.062

1.517

Nramp1 + PEI

14.31

2.862

1.908

 

2.187

 

 

0.878466

 

23.78

4.756

3.171

11.11

2.222

1.481

Figure 4c: A bar graph of the effects of PEI DNA delivery agent on luciferase activity in cells transfected with pGL3-control and pGL3-basic with Nramp1 promoter plasmid DNA.

The presence of transfection reagent PEI allows pGL3-control and pGL3-basic ('Nramp1') DNA to be delivered into the cell, as illustrated by the increase in luciferase activity in the +PEI condition using both types of promoter.

The cells used were incubated overnight in 0.5mL of DMEM growth media either with PEI or without PEI, for both pGL3 and Nramp1 plasmid DNA respectively.

As PEI is a polycation, it forms polyplexes with DNA and, therefore, condenses it and gives it a strong positive charge, allowing it to pass into the cell by endocytosis and escape endosomal/lysosomal degradation, reaching the NPC more efficiently. It was predicted that the presence of PEI DNA delivery agent in the transfection media would cause much higher luciferase activity within the cells post-transfection, which is illustrated in figures 4c, above. Statistical analysis (t-test) of the change in mean luciferase activity from 1.177 LU/μg protein, in the no PEI condition, to 24.338 LU/μg protein in the +PEI condition, for pGL3-control plasmid DNA, gave a p-value (p=<0.05) of 0.05 which is marginally significant, although the difference in luciferase activity in both conditions is much more visible than in the data collected originally, making this data more useful in this investigation.

Secondly, the increase in luciferase activity from 0.946 LU/μg protein in the no PEI condition to 2.187 LU/μg protein in the +PEI condition, for pGL3-basic (with Nramp1 promoter) plasmid DNA, gave a p-value of 0.239 which is not significant. However, the data does indicate that there is an increase in luciferase activity when the DNA is pre-incubated with PEI, compared to when it is not, suggesting that PEI causes DNA to be delivered more efficiently into the host cell nucleus.

2. The effects of H58 on transfection efficiency

Following the result of the first experiment, that PEI is necessary for DNA delivery and, therefore, for luciferase activity (transcriptional activity) within the cell, it was decided that the effect of H58 would be investigated as this has been previously researched and found to increase the efficiency of transfection (deduced by a significant increase in luciferase activity in cells post-transfection).

In this experiment, 4μL of H58 was added to the DNA-PEI and DMEM media 20mins before the samples were pipetted onto the cell plate and left to incubate overnight. The luciferase activity of the cells was then measured in a luminometer using 50μL of luciferase assay reagent, the results of which are show in figure 5a.

Figure 5a: Table outlining luciferase activity readings for each condition normalised to protein content (µg/µL) and analysed using a T-test method.

The effects of H58 on transfection (added after 0hrs)

Condition

LU/5µL

*171

LU/µL

LU/µg

Mean

Std dev

T-test (p value)

pGL3 + PEI

154.2

26368.2

5273.640

4394.7

 

4262.650

 

 

678.039

 

 pGL3 + PEI vs.

pGL3 + PEI + H(0) =

0.23160566

123.8

21169.8

4233.960

3528.3

170.7

29189.7

5837.940

4864.95

pGL3 + PEI + H(0)

123.2

21067.2

4213.440

4681.6

 

15,511.600

 

 

11771.575

 

363.5

62158.5

12,431.700

13813

737.9

126180.9

25,236.180

28040.2

Nramp1 + PEI

80.59

 

16.118

11.512857

 

12.197

 

 

4.104

 

 Nramp1 + PEI vs.

Nramp1 + PEI + H(0) =

0.00368853

59.34

 

11.868

8.4771429

116.2

 

23.240

16.6

Nramp1 + PEI +H(0)

2603

 

520.600

400.46154

 

364.615

 

34.681

2354

 

470.800

362.15385

2153

 

430.600

331.23077

Figure 5b: A bar graph of the effect of H58 on luciferase activity in cells transfected with pGL3-control plasmid DNA.

Figure 5b shows that addition of Hoechst 22358 0hrs into the transfection procedure causes an almost 4-fold increase in luciferase activity, from a mean of 4,262 LU/μg protein in the no Hoechst 22358 condition, to a mean of 15,511 LU/μg protein in the Hoechst 22358 at condition. This data gave a p-value of 0.232 which is not significant, although it shows that the transfection efficiency is affected by minor groove binding ligands, as seen in Fong et al.

Figure 5c: A bar graph of the effect of H58 on luciferase activity in cells transfected with pGL3-basic plasmid DNA.

Figure 5c shows that the addition of H58 causes increased luciferase activity in cells transfected with pGL3-basic plasmid DNA. There is an almost 30-fold increase in luciferase activity, suggesting that transfection efficiency is increased by the formation of DNA-Hoechst complexes. This data is statistically significant (p=0.037), indicating that the pGL3-basic plasmid DNA was delivered into the Raw264.7 cell nuclei more efficiently than in the no Hoechst condition, due to the membrane-permeant property of Hoechst being passed onto the DNA when DNA-Hoechst complexes are formed.

3. The effects of Heparin on transfection efficiency when added after 0hrs

The effects of heparin on transfection efficiency were the next to be investigated, with the expected outcome to be inhibition of transfection, according to the findings of Mislick and Baldeschwieler7.

In this experiment, 1μL of heparin was added per replicate well at the time of transfection with PEI (t0) and its effect on transfection efficiency was measured by change in luciferase activity inside the cells post-transfection with either pGL3-control or pGL3-basic vector.

Figure 6a: Table of luciferase readings for each condition normalised to protein content (µg/µL) and analysed using a T-test method.

The effects of heparin on transfection (added after 0hrs)

Condition

LU/5µL

*171

LU/µL

LU/µg

Mean

Std dev

T-test (p value)

pGL3 + PEI 

1172

200412.0

234.400

334.85714

 

334.238

 

 

84.359

 

pGL3 + PEI vs.

pGL3 + PEI + h(0) =

0.02060256

873.5

149368.5

174.700

249.57143

1464

250344.0

292.800

418.28571

pGL3 t + PEI + h(0

0.007

1.2

0.001

0.0035

0.043

0.049

0.195

33.3

0.039

0.0975

0.053

9.1

0.011

0.0265

Nramp1 + PEI

 

34.78

 

6.956

6.956

 

5.601

 

 

1.307

 

Nramp1 + PEI vs.

Nramp1 + PEI + h(0) =

0.02748335

27.49

 

5.498

5.498

21.74

 

4.348

4.348

Nramp1 + PEI + h(0)

0.776

 

0.155

0.3104

0.584

0.284

2.192

 

0.438

0.8768

1.409

0.282

0.5636

Figure 6b: A bar graph of the effect of heparin on luciferase activity in cells transfected with pGL3-control plasmid DNA.

The data displayed in figure 6b shows that heparin caused 99.987% inhibition of luciferase activity in cells transfected with pGL3-control plasmid DNA, indicating a significant effect on transfection efficiency as supported by the statistical analysis which returned a significant p-value of 0.021 (figure 6a).

Figure 6c: A bar graph of the effect of heparin on luciferase activity in cells transfected with pGL3-basic plasmid DNA.

When heparin was added prior to 20minute incubation with pGL3-basic ('Nramp1' in the figure) DNA, it can be seen in figure 6c that luciferase activity was inhibited by 89% when compared with cell transfected with pGL3-basic vector in the absence of heparin. Statistical analysis of this data indicates a significant change in luciferase activity between the two conditions (p=0.0275), suggesting that heparin inhibits DNA delivery into the cell, as it was added before transfection had a chance to complete, yet still exhibited a significant effect, whereas it is suspected that Hoechst has an effect when the DNA is already within the cell.

4. The effects of Heparin (added at 0, 2 and 4 hours during the transfection procedure) on transfection efficiency

Upon completing experiment 3, where heparin had been added to the DNA at the same time as PEI (0hrs/t0), it seemed that the inhibitory effect of heparin may have been occurring before the DNA had been delivered into the cell. For this reason, heparin was added at 0, 2 and 4 hours after the transfection process began in order to deduce whether heparin could still exert any effects once the DNA-PEI polyplexes had entered the cell.

For the no heparin condition, the DNA and PEI were mixed and incubated for 20mins before being added to the corresponding set of 3 replicate wells, as set out in the transfection procedure method, above.

For the t0 (0hrs) condition of this experiment, 1μl of heparin was added, per replicate well, to the DNA (pGL3-control and pGL3-basic respectively) and DMEM media at the same time as the PEI was added and transfection began.

After 2 hours (t2), 1μl of heparin was added to the next three replicate wells (for both types of pGL3 vector)

After 4 hours (t4), 1μl of heparin was added to the next three replicate wells (for both types of pGL3 vector)

Figure 7a: Table of luciferase readings for each condition normalised to protein content (µg/µL) and analysed using a T-test method.

The effects of Heparin on transfection (added at 0, 2 and 4 hours during transfection)

Condition

LU/5µL

*171

LU/µL

LU/µg

Mean

Std dev

T-test (p value)

pGL3 + PEI

281.3

48102.3

9620.460

13743.514

 

15015.429

 

 

1624.897

 

 

 

 

344.8

58960.8

11792.160

16845.943

295.9

50598.9

10119.780

14456.829

pGL3 + PEI + h(0)

0.122

20.9

4.172

6.954

 

18.943

 

 

21.660

 

pGL3 + PEI vs. pGL3 + PEI + h(0) = 0.00392426

0.104

17.8

3.557

5.928

0.771

131.8

26.368

43.947

pGL3 t + PEI + h(2)

1.491

255.0

50.992

72.846

 

79.165

 

 

46.251

 

pGL3 + PEI vs. pGL3 + PEI + h(2) = 0.00374509

2.625

448.9

89.775

128.25

0.745

127.4

25.479

36.398571

pGL3 + PEI + h(4)

141.2

24145.2

4829.040

6898.6286

 

6019.200

 

 

761.670

 

pGL3 + PEI vs. pGL3 + PEI + h(4) = 0.01949309

114.4

19562.4

3912.480

5589.2571

114

19494.0

3898.800

5569.7143

Nramp1 + PEI

9.865

 

1.973

2.1922222

 

1.361

 

 

0.721

 

 

 

 

4.353

 

0.871

0.9673333

4.149

 

0.830

0.922

Nramp1 + PEI + h(0)

0.472

 

0.094

0.1888

 

0.329

 

 

0.256

 

Nramp1 + PEI vs. Nramp1 + PEI + h(0) = 0.17485662

1.559

 

0.312

0.6236

0.433

 

0.087

0.1732

Nramp1 + PEI + h(2)

8.439

 

1.688

4.2195

 

2.291

 

 

1.823

 

Nramp1 + PEI vs. Nramp1 + PEI + h(2) = 0.31480897

1.194

 

0.239

0.597

4.115

 

0.823

2.0575

Nramp1 + PEI + h(4)

12.5

 

2.496

4.992

 

3.506

 

 

1.290

 

Nramp1 + PEI vs. Nramp1 + PEI + h(4) = 0.02276767

7.13

 

1.426

2.852

6.685

 

1.337

2.674

Figure 7b: A bar graph to show the effects of heparin on luciferase activity (when added at 0, 2 and 4 hours into the transfection process) in cells transfected with pGL3-control plasmid DNA

The data in figure 7b shows that addition of heparin to pGL3-control transfected cells at 0hrs (t0) causes a 99.874% inhibition of luciferase activity, compared to 99.473% inhibition when added after 2 hours and 59.913% inhibition when added after 4 hours. This suggests that after 4 hours, most of the transfection process would have already occurred, meaning that heparin works outside of the cell, as there was no unbound PEI left outside of the cell for heparin to compete with DNA for6,7. Statistical analysis of this data shows that the change in luciferase activity in the presence of heparin added at all three time intervals is significant, indicating that heparin does have a dramatic inhibitory effect on transfection and, therefore, providing implications for the therapeutic uses when targeting DNA to particular cells and blocking its entry to others, although it is not entirely understood how this may be achieved.

Figure 7c: A bar graph to show the effects of heparin on luciferase activity (when added at 0, 2 and 4 hours into the transfection process) in cells transfected using pGL3-basic plasmid DNA

For cells transfected using pGL3-basic ('Nramp1' in figure 7c), the addition of heparin at 0 hours causes 76% inhibition of luciferase activity compared with a 68% increase in luciferase activity when added after 2 hours and an even greater increase of 158% when added after 4 hours. These results suggest that heparin is increasing transfection efficiency when added after 2 and 4 hours, which is not what they were expected to show. It was predicted that heparin would cause the most inhibition when added at 0 hrs, as all PEI and DNA would be outside of the cells, allowing heparin to bind PEI, competing with the DNA which forms polyplex with PEI which are able to be transported into cells. One explanation for the results obtained could be that there was less DNA in the no heparin ('Nramp1 + PEI') condition and, therefore, less entered the nucleus of the cells overall, causing lower luciferase activity. However, the protocol used for each condition was kept constant, so this is unlikely.

Discussion

The primary aim of this experiment was to establish the role of polyethylenimine (PEI) as an effective DNA delivery agent by investigating its effect on transfection efficiency as measured by luciferase activity within cells (fig. 4c). The results for the pGL3-control DNA illustrated that, whilst some luciferase activity was measure in the absence of PEI, there was a significant increase in transfection efficiency when PEI was added to the DNA, increasing luciferase activity by 20-fold. The difference in transfection efficiency was not as great in the case of pGL3-basic DNA, but still showed an increase of ~2-fold (fig. 4b). These results are in accordance with previous findings; that PEI had formed DNA-PEI polyplexes, which bind to HSPG on the cell surface and induce endocytosis of the PEI-bound DNA5,6,7.

The subsequent experiment was undertaken to investigate whether DNA minor groove binding ligand Hoechst 22358 had an effect on the efficiency of transfection when added at 0hours into the process. The results showed that Hoechst caused a 4-fold increase in transfection efficiency when using pGL3-control vectors (fig. 5b), compared to an increase of 30-fold when using pGL3-basic vectors (fig. 5c), supporting previous findings by Fong et al10. In order to follow up these findings, it would be interesting to carry out the same experiment adding different concentrations of Hoechst 22358 in order to see whether the level of luciferase activity is altered proportionally. Alternatively, H58 could be added at different time intervals (as with the heparin in experiment 4), in order to establish whether the DNA minor groove binding ligand was exhibiting its effect pre- or post-DNA delivery into the cell or at different concentrations to explore the toxicity associated with minor groove binding ligands which is often the property which gives viral vectors the upper hand for DNA delivery in therapeutic techniques.

The mechanism by which transfection efficiency is increased by H58 has been investigated by numerous groups, resulting in an abundance of possibly explanations. Previous research suggests that H58 upregulates expression of DNA once it is inside the cell by a pathway in which, Fong et al. state, "Hoechst may target complexed plasmid DNA to transcriptionally active sites on nuclear chromatin"10, paving the way for potential new therapeutic strategies by which targeting of transfected DNA may increase expression in host cells.

The recruitment of transcription factors by Hoechst within the nucleus can be explained by the formation of TATA box-binding protein (TBP)-TATA box element complexes as investigated using gel mobility shift assays9, a technique which allows DNA-protein complex formation to be quantified, and complexes contained within a [32P]-labelled 24-bp oligonucleotide. The presence of Hoechst causes an increase in the formation of transcription factor/TBP-TATA box element complexes which are required for recruitment of RNA polymerase II in transcription9. The mechanism by which this occurs has not yet been fully elucidated, emphasising the need for further investigation into how this occurs in order to reap potential therapeutic benefits of the use of H58 in the future9.

Another explanation for the increase in transfected gene expression is an alteration in rotational positioning of the DNA around the nucleosome core as explored in the study by Brown and Fox (1996)13. Formation of a nucleosome causes DNA of 145bp in length to be wrapped around 1.8 times13, bending the helix so that specific regions of its sequence are faced inwards, towards the protein surface of the histone octamer core13.

One type of sequence positioned towards the protein surface within the packaged nucleosome are those rich in AT bases, which are the preferred binding sites for minor groove binding ligands such as H5813. It was found that Hoechst binds to the AT-rich sequences which are facing inward (towards the protein) causing a 5 base pair shift in the positions of maximal cleavage, rotating the DNA by 180° and causing regions facing away from the protein to face toward it and vice versa, minimizing electrostatic repulsion between the positively charged groups on the histone core and cationic ligand termini13. This rotation may also cause the exposure of specific binding sites for transcription factors, possibly providing an explanation for the increase in luciferase activity (by increased luciferase gene expression).

Alternatively, it has been suggested that the bendability of the DNA may be reduced by the binding of Hoechst and other similar DNA minor groove binding ligands13. This would cause the DNA to bend less or even the wrong way, which would explain the rotational change in the DNA position in relation to the histone core, as the DNA and histone are required to remain in contact13. Although only low levels of occupancy were required to cause DNA rotation, ligand was found to still be bound at TTAA and TATA sites which suggests Hoechst has increased specificity and affinity for these sites, compared to the other ligands used in the research13. In addition to rotational movement, the DNA wrapped around the nucleosome core was also found to be dynamic, with the binding of ligand causing it to move along the surface of the nucleosome as well as rotating about its axis. This mechanism could explain the large scale change in DNA conformation in some of the conditions used in the study13.

Recently, it has been suggested that minor groove binding ligands, such as H58 used in this experiment, have implications in treatment of cancer as anti-tumour agents14. The effect of Hoechst on gene expression has been investigated in Saccharonmyces cerevisiae (yeast) in single-gene analysis studies using microarray and gel-based methods to investigate positive and negative effects of minor groove binding ligands on gene expression. For example, the sequence ATTTT has been linked to an alteration in gene expression when ligand binds to it14.

The inhibitory effect of heparin on transfection efficiency was then investigated, showing that, when added at t0 (0 hours into the transfection), it inhibited transfection by as much as 99% (fig. 6a/6b). This is due to its negative charge density, allowing it to interact with the DNA delivery agent PEI, which has a net positive charge, displacing bound DNA from the PEI and preventing formation of polyplexes6. This competition between DNA and Heparin means that, depending on the extracellular concentration of heparin, a lot of the PEI molecules will be free of DNA, reducing the number PEI-DNA polyplexes which could bind to HSPG on the cell surface7. This is a potential cause of less DNA molecules successfully reaching the nucleus or being expressed, which would explain the reduction in luciferase activity seen in figure 6a, although there is not much research into whether DNA displacement PEI can directly disrupt DNA entry into the cell via HSPG6,7.

When added later in the transfection process, the effect of heparin on transfection efficiency was not nearly as pronounced. This is due to the formation of PEI-DNA polyplexes earlier in the process which can pass into the cell via HSPG, avoiding the effects of heparin when added later on. This potentially allowed much more DNA to be transported into the cell before the heparin was added, explaining the effect on luciferase activity in figure 6b/6c and supporting the evidence that heparins action is extracellular7.

The results of this study support the findings of previous research but also raise some questions about the future of research in this area. However, the principle weakness in this study is that of the validity of the measurement of transfection efficiency. This could be improved upon in future studies by use of DNA in situ hybridization15, which can be used to directly detect specific DNA sequences within the cell, providing a more accurate look at expression of particular DNA.

Conclusion

The results of this investigation support the findings of previous research into the mechanism of efficient transfection and also help further our understanding of the implications for use of such mechanisms in therapeutic techniques, such as gene therapy. This is highlighted by research into novel DNA delivery vehicles, such as PEI, which helps to increase transfection efficiency and also by way of DNA minor groove binding ligands, such as H58 which has been seen to increase transfection efficiency and remain relatively non-toxic at low concentrations, making it, and other compounds like it, seem realistic alternatives to viral vectors in the not so distant future. Although further exploration is required in order to elucidate the exact mechanism by which Hoechst exhibits its effects, further investigation may lead to the discovery of a safe and efficient transcriptional enhancer which could be used to combat diseases such as cancers, which are growing evermore common.

References

1. Wagstaff, K.M. and Jans, D.A. (2007) Nucleocytoplasmic transport of DNA: enhancing non-viral gene transfer. Biochem J. 406; 185-202.

2. Godbey, W.T., Wu, K. and Mikos, A. (1999) Polyethylenimine and its role in gene delivery. Journal of controlled release. 60; 149-160.

3. Terry, L.J., Shows, E.B., Wente, S.R. (2007) Crossing the nuclear envelope: hierarchical regulation of nucleocytoplasmic transport. Science. 318;1412-6.

4. Boussif, O., Lezoualch, F., Zanta, MA., Mergny, MD., Scherman, D., Demeneix, B. and Behr, J.P. (1995) A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: Polyethylenimine. Proc Natl Acad Sci USA. 16; 7297-7301.

5. Thomas, M., Klibanov, M. (2003) Non-viral gene therapy: polycation-mediated DNA delivery. Appl Microbiol Biotechnol. 62; 27-34.

6. Ruponen, M., Yla-Herttuala, S., Urtti, A. (1999) Interactions of polymeric and liposomal gene delivery systems with extracellular glycosaminoglycans: physiochemical and transfection studies. Biochimica et Biophysica Acta. 1415; 331-341

7. Mislick, K.A. and Baldescwieler, J.D. (1996) Evidence for the role of proteoglycans in cation-mediated gene transfer. PNAS. 93; 12,349-12,354.

8. Guan, Y., Shi, R., Li, X., Zhao, M., Li, Y. (2007) Multiple Binding Modes for Dicationic Hoechst 33258 to DNA. J Phys Chem B. 111; 7336-7344

9. Zhang, X., Kiechle, FL. (2003) Hoechst 33342 alters luciferase gene expression in tranfected BC3H-1 myocytes. Arch Pathol Lab Med. 9; 1124-1132.

10. Fong, S., Liu, Y., Heath, T., Fong, P., Liggitt, D., Debs, R.J. (2004) Membrane-permeant, DNA-binding agents alter intracellular trafficking and increase the transfection efficiency of complexed plasmid DNA. Mol Ther. 4; 706-718.

11. Dervan, P.B. (1986) Design of sequence-specific DNA-binding molecules. Science. 232(4794); 464-471.

12. Wong, S.S., et al. (1994) Transcriptional regulation of differentiation, selective toxicity and ATGCAAAT binding of bis-benzimidazole derivatives in human melanoma cells. Biochem Pharmacol. 47; 827-837.

13. Brown, P.M. and Fox, K.R. (1996) Minor groove binding ligands alter the rotational positioning of DNA fragments on nucleosome core particles. J. Mol.Biol. 262; 671-685.

14. Hart, S.N. and Eckdahl, T.T. (2006) Effect of Hoechst 33258 on gene expression in yeast. The FASEB Journal. 20; A73.

15. Wilkinson, D.G. (1999) In situ hybridization: a practical approach. Oxford: Oxford University Press.

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