20.109(S09): Transfection (Day5)

Introduction

Welcome back to Module 2 - it's been a while! Between the foundational background you gained in the first half of the module, and the well-deserved break, I hope you're ready to head full force back into lab work and analysis. Today you will revisit MES cell culture, transfecting these cells with DNA and/or RNA for two experiments. First, you will confirm that our MES utilize RNAi pathways, and compare the effectiveness of different siRNA designs against a report (luciferase). To do this, today you will simultaneously transfect MES with a luciferase gene and with an siRNA targeting luciferase (along with performing suitable controls), and next time you will test for luciferase knockdown using a luminescence assay. In the second experiment, you will transfect MES with an siRNA against an actual mouse gene. Over the next week you will perform a genome-wide expression assay and assess direct and indirect affects of the mouse siRNA.

Let's start by learning more about luciferase, which is an enzyme that oxidizes its substrate, luciferin. The product, oxyluciferin, emits light in the blue range of the visible spectrum, 440-479 nm. The oxidation reaction is fundamentally different from another light emitting reaction that you are now intimately familiar with, fluorescence. During fluorescence, absorbed light is re-emitted by the excited fluor as light of a longer wavelength (this is called a Stokes shift); in the case of GFP, absorbed blue light is emitted as green light, ~ 505 nm. Interestingly our luciferase source, Renilla reformis, has both a luciferase-luciferin pair as well as GFP. Consequently the oxidation reaction can lead to oxyluciferin luminescence and then to fluorescence, through energy transfer to GFP, giving the soft corals a blue-green glow.

Luciferin-luciferase pairs are widely used in nature for courtship, camouflage or baiting. Fireflies (also known as lightning bugs or more technically Photinus pyralis) use an ATP-requiring luciferin-luciferase pair and emit species-specific patterns of light as part of their mating ritual. Bioluminescent bacteria (such as Vibrio harveyi) can be found in symbiotic relationships with marine organisms. The fish give such bacteria a suitable home and in return the bacteria emit light to give the fish “night vision,” or to mask the fish’s shadow, effectively cloaking them from their prey. The usefulness of such luciferin/luciferase pairs was not lost to researchers who began isolating and sequencing luciferase genes from different organisms in order to clone them into useful vectors, such as the one we’ll use today.

Unexpectedly, there is little primary sequence similarity for luciferases from different organisms. This finding will be used to our advantage as we target mRNA from the Renilla luciferase gene for destruction while using the product of the firefly luciferase gene as an unaffected control. On the plasmid we will use, each gene is controlled by a strong constitutive promoter, leading to high expression levels of both luciferases when the plasmid enters a mammalian cell. Other plasmid features that you will be familiar with from experimental module 1 include an antibiotic resistance gene (in this case against the antibiotic ampicillin that serves as a selectable marker in bacterial cells), a bacterial origin of replication, and multiple restriction sites that can be used for cloning.

In many ways, the assay for luciferase activity is a "standard" assay. The enzyme and substrate must react for a defined time and the amount of product gets recorded. What's a little unusual is that in this case the measured product is light and the light can be quantified using a luminometer. Today’s reactions are slightly more advanced than standard ones in that you’ll perform two sequential measurements. The first reaction is initiated with Beetle luciferin, a substrate for firefly luciferase. The light produced, called a “flash reaction” because it does not persist, will be measured for 10 seconds and then a reagent to stop the reaction will be added. The stop reagent also contains the substrate to initiate the second reaction. Coelenterazine reacts with Renilla luciferase in a “glow reaction,” which decays more slowly, but you will measure it for precisely 10 seconds so the lumens emitted can be compared to those from the firefly luciferase. There are several attractive aspects to these reactions including the assay’s low cost, high speed, great sensitivity (to attomolar amounts of luciferase, that’s 10^-18th!) and wide dynamic range, linear through 7 orders of magnitude. It is also beneficial that there is no endogenous luciferase-luciferin pair in most experimental organisms.

There are two parts to today’s lab. Half the class will begin in the TC facility transfecting MES cells with the luciferase reporter plasmid and/or siRNAs. The other half of the class will begin by testing some control extracts from transfected cells to become familiar with the luciferase assay and statistical analysis of the data that is generated. Midway through the lab period, the groups will switch places so everyone will have an opportunity to perform both protocols.

Protocols

Part 1: Sign-up for mouse gene

Please select one mouse gene to test from the list below. Up to two groups per day will test the same gene - which will allow us to do some neat controls later, as well as give us a sense of our data's reproducibility - but try to make sure that each gene is represented at least once first.

You can learn more about mouse genes at Mouse Genome Informatics.

Gene Name	Protein Function	T/R Group 1	T/R Group 2	W/F Group 1	W/F Group 2
Mpg (N-methylpurine-DNA glycosylase)	glycosylase involved in DNA repair	Grey	Yellow	Orange
Trp53 (transformation related protein 53)	DNA binding, cell cycle/apoptosis; no transcriptional activity in ES cells	Blue	Green	Pink
Polr2d (polymerase (RNA) II (DNA directed) polypeptide D)	non-essential subunit of RNA polymerase
Tada2L (transcriptional adaptor 2 (ADA2 homolog, yeast)-like)	transcriptional activator	Orange	Purple	Red
Nanog (homeobox)	ES cell-specific transcription factor	Red	Pink	Purple

Part 2: Transfection

DNA can be put into mammalian cells in a process called transfection. Mammalian cells can be transiently or stably transfected. For transient transfection, DNA is put into a cell and the transgene is expressed, but eventually the DNA is degraded and transgene expression is lost ("transgene" is used to describe any gene that is introduced into a cell). For stable transfection, the DNA is introduced in such a way that it is maintained indefinitely. Today you will be transiently transfecting your cultures of mouse embryonic stem cells.

There are several approaches that researchers have used to introduce DNA into a cell's nucleus. At one extreme there is ballistics. In essence, a small gun is used to shoot the DNA into the cell. This is both technically difficult and inefficient, and so we won't be using this approach! More common approaches are electroporation and lipofection. During electroporation, mammalian cells are mixed with DNA and subjected to a brief pulse of electrical current within a capacitor. The current causes the membranes (which are charged in a polar fashion) to momentarily flip around, making small holes in the cell membrane through which the DNA can pass.

The most popular chemical approach for getting DNA into cells is called lipofection. With this technique, a DNA sample is coated with a special kind of lipid that is able to fuse with mammalian cell membranes. When the coated DNA is mixed with the cells, they engulf it through endocytosis. The DNA stays in the cytoplasm of the cell until the next cell division at which time the cell’s nuclear membrane dissolves and the DNA has a chance to enter the nucleus.

Two days ago, cells were prepared for you at a density of 4-5 X 10^5 cells/well in all six wells of two six-well plates.

A schematic of your experiment is shown below. You can see that most of the samples are controls! This is a very common feature of scientific investigations, because there are many possible explanations for a given outcome, some of which can be ruled out (or confirmed, alas) using controls.

All manipulations are to be done with sterile technique in the TC facility. In addition, since you will be working with RNA, it is important to wear gloves whenever you are handling the transfection reagents. This will protect them from degradative enzymes on your fingers.

Timing is important for this experiment, so calculate all dilutions and be sure of all manipulations before you begin.

Lipofection scheme

For each lipofection you will need

• Carrier: 3 ul Lipofectamine 2000 in 50ul OptiMEM (50 ul is the final volume, so don't forget to subtract the volume of Lipofectamine)
• DNA: 20 ng of psiCHECK2 in 50 ul OptiMEM (final volume) and/or
• luciferase siRNA: 10 pmoles in 50 ul OptiMEM (final volume) and/or
• mouse gene siRNA: 100 pmoles in 50 ul OptiMEM (final volume)

Each of the 12 sample wells will receive ~100 μL of a solution containing equal parts carrier and diluted nucleic acid(s). The 12 wells comprise 6 unique samples tested in duplicate.

1. Prepare enough carrier for 12.5 lipofections. Let the diluted lipofectamine sit in the hood undisturbed for at least 5 minutes but not more than 30.
2. All the lipofections will be done in duplicate with 20 ng of DNA/well and/or 10-100 pmoles of siRNA/well. Prepare each nucleic acid cocktail with enough material to perform both replicates (100 μL), in an eppendorf tube. The following table may be helpful for your calculations.

Tube	[DNA] stock	Volume DNA	[siRNA] stock	Volume siRNA	OptiMEM
No DNA (2 replicates)	---	---	---	---	100 ul
plasmid only (2 replicates)	20 ng/ul		---	---
scrambled siRNA only (1 replicate only!)	---	---	10 pmol/ul
mouse gene siRNA only (1 replicate only!)	---	---	10 pmol/ul
plasmid+siRNA (validated) (2 replicates)	20 ng/ul		10 pmol/ul
plasmid+siRNA (scrambled) (2 replicates)	20 ng/ul		10 pmol/ul
plasmid+siRNA (experimental) (2 replicates)	20 ng/ul		10 pmol/ul

3. Add an equal volume of diluted carrier to the six nucleic acid(s) dilutions you prepared above, and pipet up and down to mix each now 200 μL solution.
4. Incubate the DNA/RNA/lipofectamine cocktails undisturbed at room temperature for 20 minutes. During this time, aspirate the media from the cells in your 6-well dishes, wash the wells with 2 ml PBS from a 10 ml pipet, then put 1 ml of fresh Pre-transformation Media on the cells, dispensed from a 5 ml pipet.
5. After the 20 minute incubation is over, use your P200 to add 95 ul of the appropriate DNA/RNA/lipofectamine complexes to each well. Since the carrier can be toxic to cells it’s a good idea to gently rock the plate back and forth after each addition.
6. Return the plate to the 37° incubator.
7. One of the teaching faculty will complete the transfection protocols by performing the following steps:

• Plates will be incubated overnight @ 37°C, 5% CO2.
• Tomorrow, the media and transfection reagents will be aspirated and replaced with 3ml fresh JI growth media with serum and antibiotics.
• Plates will be incubated overnight @ 37°C, 5% CO2.

Next time you and your partner will collect cells from today’s transfections to analyze their luciferase activity and isolate total RNA for microarray analysis.

Part 3: Practice luciferase reactions

In the main teaching lab you will have some cell lysates to study. The precise identity of these samples is not important. Rather, you should use them to familiarize yourself with the mechanics of the dual-luciferase assay as well as the particulars of the resulting data analysis. Before starting your reactions, you will hear a short lecture on statistics.

General considerations

• Assays should be performed without gloves since these may generate static electricity that will be detected by the luminometer.
• All reagents must be at room temperature.
• PBS can be used to dilute lysates that give readings beyond the upper range of the luminometer (“>9999”). Needless to say the dilution factor must be taken into account when analyzing your data so keep track.
• Do not pipet less than 10 ul of lysate to each reaction or for each dilution since the error in pipeting smaller volumes may confound your data analysis.
• Do not make any additions to the eppendorf while holding it directly above the luminometer. Airborn droplets may fall into the machine.
• Keep the luminometer’s sample lid closed as much as possible. The clicking noise you hear when it’s open should remind you to do this.
  

Protocol

1. Add 50 ul of LARII to a series of three eppendorf tubes. LARII has Beetle luciferin, the substrate for firefly luciferase.
2. Add 10 ul of cell lysate to the first tube only. Pipet up and down several times to mix then close the cap. The reaction between any firefly luciferase and LARII will measurably decay after only 2 minutes so it is important to collect your data relatively quickly.
3. Place the eppendorf into the Turner Luminometer 20/20 and press the green “GO” button. One of two things will happen. Either the machine will collect a 10 second record of the light from your sample (in which case you should write down the number then proceed to step 4), or it will inform you that the sample is out of range (>9999) in which case you should return to step 2, trying a 1:10 dilution of your lysate.
4. Once the first measurement has been made, add 50 ul of the “Stop and Glo” reagent, which will both quench the first reaction and also initiate the second. You should do this before starting any other LARII reactions with other lysates.
5. Measure the light produced from the reaction of the Renilla luciferase just as you did for the firefly luciferase.
6. Repeat for the next two samples. Try to be consistent about how long you wait between pipetting the reagent and measuring the signal.
7. Post your data on today's Talk page (see more info below).

Analysis

• Download the following Excel file as a framework to carry out the basic statistical manipulations we discussed. The file is modified from one originally written by Professor Bevin Engelward.
• Compare the pooled class data for the three different lysates tested today following the procedures below. You and your partner can hand in one copy of your Excel sheet as part of your notebook.

1. Open a second Excel worksheet. Here, convert any luminescence data you collected into ALU/ul of lysate where ALU is an abbreviation for "arbitrary luminescence units."
2. In the RNAi experiment you have performed, the firefly luciferase should be unaffected by any of the siRNAs and thus can be used to normalize for transfection efficiency in each well. With the data you collected today, express each Renilla luciferase measurement as a fraction of the firefly luciferase value. Post both your raw data and the ratios to today's Talk page. In your notebook, answer the following:
   • What does a value less than one tell you?
   • If you were to plot the ratios on an X-Y axis, would would a four-fold increase and a four-fold decrease be visually equal?
3. Combining parts 1 and 2, present three replicates of normalized data (your own and from two other groups) in a well-labeled bar graph with error bars reflecting the 95% confidence intervals, and brackets/asterisks indicating differences significant at the 95% confidence level. There is no need to include a title and caption for this figure.

For next time

1. The final draft of your Module 1 research article is due by 11 AM next time. Please name your files according to the following convention: Firstinitial_Lastname_LabSection_Rev-Mod1.doc and email them to 20109.submit@gmail.com

Reagents list

• Pre-transfection Medium
  • J1 growth medium (see Day 1), less Pen/Strep and non-essential amino acids

• Validated Renilla luciferase siRNA (all siRNA stocks are 10 pmoles/ul)
  • GGC.CUU.UCA.CUA.CUC.CUA.C dT.dT
  • dTdT CCG.GAA.AGU.GAU.GAG.GAU.GA
• Scrambled siRNA, also called non-targeting siRNA#2 from Dharmacon
  • UAA.GGC.UAU.GAA.GAG.AUA.C dT.dT
  • dTdT AUU.CCG.AUA.CUU.CUC.UAU.GA

• LARII and Stop&Glo
  • Promega dual-luciferase assay reagents