DNA can be put into mammalian cells in a process called transfection. If you wanted to make a mouse cell fluoresce green, you could transfect it with DNA carrying the EGFP open reading frame, a promoter directing transcription of EGFP and a signal sequence for polyadenylation of the mRNA. The promoter tells the cell that the EGFP sequence should be transcribed by RNA polymerase. The polyadenylation sequence assists in the export and stability of the mRNA so that it gets translated by the ribosome. The coding sequence tells the ribosome which amino acids should be joined together.
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 that the DNA can pass through.
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.
Today you will lipofect several DNA samples into your mouse embryonic stem cells. As a positive control, you will transfect one sample with a plasmid encoding full-length EGFP. This plasmid will cause any transfected cells to fluoresce green. Next time we will measure fluorescence of your positive control to assess the success rate of the transfection.
You will also be transfecting two experimental plasmids, one of which you have been constructing for weeks. The EGFP coding sequence on these plasmids is truncated at either the 5’ or 3’ end of the gene. Cells expressing these truncated EGFPs should not fluoresce green. The plasmids provide a wonderful tool for studying recombination since a cell will fluoresce green if it has been transfected with both plasmids and has recombined the genes to regenerate a full-length EGFP. Finally, you will be using the truncated EGFP plasmids to investigate the effect of double strand breaks on the frequency of recombination.
Elaborate mechanisms for coping with DNA breaks have evolved since these forms of DNA damage are so dangerous for the cell. You should (re)familiarize yourself with these mechanisms by (re)reading the excellent review by Thomas Helleday that you can find in the References section of the Module 1 frontpage. You should also check out the animations of repair mechanisms that are linked there. These animations were made by Justin Lo, a class of '08 Course 20 student and a former UROP student in Professor Engelward's laboratory.
One kind of DNA damage with particularly catastrophic consequences for the cell is double stranded breaks. The broken ends of the DNA must be correctly repaired (literally here re-paired) without loss of any encoded information. The model for such repair is called to the Szostak model, named after the person who first described it. According to this model, illustrated below, a gap in one chromosome is repaired through an interaction with its homologous chromosome. The repair first requires “homology searching” and invasion of the gapped DNA (blue) into the undamaged copy (red). The undamaged DNA serves as a template for missing sequences, which are copied and then resolved as indicated by the open triangles on the figure below. Resolution can lead to exchange of sequences flanking the original double strand break, resulting in recombination of genetic information. Alternatively the integrity of the original genetic material is preserved when the repaired strands resolve without crossover.
Szostak model for double strand break repair
Different, and far simpler means of repair are possible if the broken ends of the DNA can be held together, either through base pairing of the overhangs or through the chromatin structure surrounding the damage. Your investigation today will assess recombination rates for different DNA lesions and conditions for homology searching.
While half of the class works in tissue culture, the other half will carry out the data analysis associated with your CometChip assay.
Part 1: CometChip Data Analysis
While you were out, there was a significant amount of work done to complete your CometChip assay (Thank you, Isaak!). Below is a summary of the steps that were completed for you and a synopsis of the relevant data analysis. You'll recall that Prof. Engelward has already gone over how to approach this type of data from a statistical point of view -- now it is your turn to flex your biostatistics muscles (you'll get another chance on M1D7 when analyzing your flow cytometry data).
- Once the MSCs that you seeded last time settled into the microwells of the CometChip, the media was aspirated from each well of the bottomless 96-well plate.
- The 96-well plate was removed and the CometChip gel was rinsed gently with PBS to remove cells not captured in microwells.
- After confirming cells were correctly loaded by examining under the TC microscope, low melting point (LMP) agarose was added to the top of the gel to trap the cells within the microwells. Note: LMP agarose melts at approx. 42C and cools very quickly so the cells are not exposed to molten agarose!
- Extra LMP agarose was removed from the edge and the gel was cut so that empty wells were tossed in the biohazard trash.
- Next, each small section of gel was placed on ice and covered with a bit of PBS.
- The gels were transported to the Koch Institute irradiation facility and exposed to 0, 2, 4, 6, 10, and 20 Gy of radiation (one gel per radiation level -- each of you prepared one strip of gel).
- After irradiation the gels were incubated overnight at 4C in lysis buffer to break open the cells and unwind the DNA.
- The next day gels were rinsed with PBS and then adhered to the bottom of a gel electrophoresis box using double sided tape.
- After equilibration in 4C alkaline running buffer, the damaged DNA was electrophoresed at 1V/cm at 4C for 30 min to create the comet tails.
- The gels were then neutralized with 3 washes of 0.4M Tris, pH 7.5 and stained with SybrGold DNA stain at 1:1000 at room temperature for 20 min.
- Finally, the microwells were imaged using a XXXX fluorescent microscope.
- Here is where I need to decide how much Matlab (if any) analysis will be done by the students
Part 2: Lipofection
In anticipation of your lipofection experiment, one of the teaching faculty plated 1x105 cells in 16 wells of a pregelatinized 24-well dish 24 hours ago. A special media formulation without antibiotics was used.
A schematic for your experiment is shown below. We will discuss in class different things we might test, and we will use wells labeled 'A' 'B' and 'C' to test a parameter that might affect the frequency of inter-plasmid homologous recombination.
All manipulations are to be done with sterile technique in the TC facility.
Timing is important for this experiment, so calculate all dilutions and be sure of all manipulations before you begin.
For each lipofection you will need
DNA: 0.1 μg (of Δ3) and/or 0.1 μg (of Δ5) in 50 ul OptiMEM
Carrier: 2.5 μL Lipofectamine 2000 in 50 μL OptiMEM
- 1. Dilute enough carrier for 16.5 lipofections. Let the dilution sit in the hood undisturbed for at least 5 minutes but not more than 30.
- 2. For the lipofections to be done just once, you’ll need 50 ul of DNA in OptiMEM. Use the table below to help you calculate the appropriate volumes to use in your experiment.
|| [DNA] stock
|| Volume DNA
|| Volume OptiMEM
|| 0.05 ug/ul
|| 0.1 ug
|| 0.05 ug/ul
|| 0.1 ug
|| 0.05 ug/ul
|| 0.1 ug
- 3. For the lipofections to be done in triplicate, you’ll need a total of 150 ul of DNA diluted in OptiMEM. By making one lipofection cocktail that is later divided between replicates, you can be confident that each replicate was treated identically. The basic protocol is to do each lipofection with 0.1 ug of ∆5 and an equal, greater, or lesser amount of ∆3.
|| [DNA] stock
|| Volume DNA
|| Volume OptiMEM
|| 0.05 ug/ul for Δ5|
0.05 ug/ul for Δ3
| 0.1 ug of Δ5 and a variable # ug of Δ3
- 4. You should now have at least 7 eppendorf tubes, four with 50 ul of OptiMEM +/- DNA and three (or four if you've done an additional variable) with mixtures of Δ5 and Δ3 DNA in a volume of 150 ul. Add an equal volume of diluted lipofectamine to each eppendorf (i.e., 50 ul if the tube has 50 ul). Pipet up and down to mix.
- 5. Incubate the DNA and lipofectamine cocktails at room temperature for 20 minutes. To allow the DNA/carrier complexes to form, it is important that you do not disturb the tubes during this incubation. During this time, aspirate the media from the cells in your 24-well dish, wash the wells with 0.5 ml PBS, then put 0.5 ml of fresh media on the cells. The PBS and media can be aliquoted with a 5 ml pipet.
- 6. After the 20 minute incubation is over, use your P200 to add 95 ul of the appropriate DNA:lipofectamine complexes to each well. Since the carrier is quite toxic to the cells it’s a good idea to gently rock the plate back and forth after each addition.
- 7. Return the plate to the 37°C incubator.
- 8. Tomorrow, one of the teaching faculty will remove the lipofection media from your cells and will replace it with 1 ml of fresh media. Approximately 48 hours after performing the lipofection, you and your partner will collect the cells and analyze their fluorescence by FACS.
BEFORE NEXT TIME
Sign up for time at the FACS here.
Groups will have to stagger their arrival at lab in order to keep the number of groups and samples at the FACS facility manageable.