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Summary Experimental Cell Biology II
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- Vrije Universiteit Amsterdam (VU)
Experimental Cell Biology II
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Wednesday, 5 October 2022Basic Techniques
CELL PREPARATION
The breakup of tissue to obtain cells can be done by a grinder/blender, osmotic shock, high
pressure (through small outlet), or via detergent/enzyme treatment (break bacterial cells).
The approach strongly depends on the tissue and cell type
DNA TECHNIQUES
DNA isolation
To isolate DNA from cells, a cell extract needs to be prepares. There is removal of the other
cell components (proteins, lipids, RNA) by treatment with detergent or organic solvent.
After, the DNA is precipitated with alcohol (ethanol or isopropanol) and dissolved in buffer
solutions.
Purity of the DNA is checked by agarose gel electrophoresis. A porous gel from agarose
and water is used to separate the DNA fragment. In the elective field, negatively charged
DNA moves. The mobility of the DNA is dependent on the length. In the electrophoresis, the
gel is stained with DNA-binding dye. This shows a pattern of fragments of different sizes. If
needed, the suitable fragment can be cut out.
DNA manipulation/cloning
Purified DNA manipulation knows different elements.
Restriction digest —> splitting by enzymes, which cut at specific sites. Also known as
restriction endonucleases as they cut between the 3’ and 5’ end.
Ligation —> connection to another DNA fragment. This process needs DNA ligase
Expression —> transcription in mRNA< translated to yield proteins. This process needs
RNA polymerase and ribosomes and is usually done within the suitable host cell
Amplification —> amplification to have enough material for the application. This is
done via PCR (polymerase chain reaction).
Restriction digest and ligation are key elements of DNA manipulation
Polymerase Chain Reaction (PCR)
PCR needs a DNA fragment to amplify, nucleotides, buffer, DNA polymerase, and a PCR
machine. The PCR has multiple steps
1. Heat is used to separate the DNA strands
2. DNA primer binds (lower T)
3. DNA polymerase makes a new strand
This PCR cycle is repeated 20-40 times for enough replication
Reverse-Transcription PCR (RT-PCR) is a modified version of PCR that can amplify RNA.
This technique can be used to investigate mRNA in a given cell. To induce RT-PCR, reverse
transcriptase is added to synthesis DNA from mRNA. After, standard PCR is performed
Real-time PCR (qPCR) is used to amplify an at the same time quantify DNA. DNA can be
quantified by the binding of fluorescence dyes. qPRC can occur in two ways
1. The dyes bind to double strand DNA, so when more DNA is produced, there is a
higher fluorescence intensity.
2. In qPCR, there is a DNA probe with the dye, and there is a low fluorescence
intensity. The probe binds to DNA and DNA polymerase splits the probe, allowing
the fluorescence intensity to go up
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Development of PCR includes the potential of PCR without thermocycling. This could be
used for medical diagnostics in the field.
Example: NASBA for detection of viral RNA
1. Reverse transcriptase makes DNA from RNA
2. RNAse H degrades the RNA bound to DNA
3. RNA polymerase makes new RNA, which is used for step 1
Mutagenesis
Mutagenesis introduced point mutations into DNA. This can be
used to investigate the effect on a proteins, which role does the
changed base/amino acid play?
Site directed mutagenesis uses a primer/oligonucleotide with a
changed base. The DNA polymerase makes DNA with mutation,
which is afterwards inserted into the host
Sanger method
The Sanger Method replicated the DNA strands. This includes the
percentage of replication-stopping fluorescent nucleotides. This
method is used to identify the DNA. The DNA is dentaured,
whereafter a primer is added and a new nucleotide can be formed
PROTEIN TECHNIQUES
Protein purification
Protein purification allows to investigate the protein without contamination of other cell
components. Protein purification is done via column chromatography. The protein sample
passes through the column, which is filled with porous material. The protein speed will
depend on the type of protein, and this method is very powerful. Column chromatography
can be done in two ways
1. Gel filtration —> usage of column material with narrow pores. Separation is
dependent on protein size. The large proteins elute first
2. Ion exchange —> column material with charged surface is used. Separation is
dependent on protein charge. Proteins without charge or with the same charge as
the column elute first
Protein presence, purity, and size is detected via polyacrylamide gele electrophoresis
(PAGE), which can separate protein, determine their size, and purity. A negatively charged
detergent (SDS) is added to the protein sample. This allow the negative charged proteins to
move to the positive electrode (Anode). When staining the proteins, they will become visible
as bands
Protein production/localization
Fluorescent protein labels are used to detect protein production. There are many types of
labels available, including staining whole cells, DNA, and proteins. Fluorescent protein
labels are a useful tool in cell biology, as they can use the fluorescent protein to one specific
protein. This technique is very powerful in combination with microscopy.
GFP fusions allow protein localisation by ,microscopy. GFP fuses to the import sequence of
mitochondria, allowing to detect the location and dynamic of mitochondria is the cells.
Mutations of GFP give many spectral variants. Also, more tan one protein can therefore be
followed and localised
Protein interaction partners (co-immunoprecipitation) can also be investigated used to find
cellular binding partners of proteins of interest. A cell/organelle extract is made and an
antibody is added against the protein of interest. After, it is tested which other proteins are
precipitated.
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KNOCKOUT MOUSE
A knockout mouse is a laboratory mouse of which one gene is knocked out. This allowing
to gain information on what the gene is good for. Knockout mice are often used to
determine the function of medically relevant genes/proteins. It is a simple, but powerful
approach.
Knockout mice can show macroscopical and microscopical alterations, and are mainly
used in studies involved in obesity and cancer.
A knockout mouse is made by culturing embryonic stem cells. These are transfected with
DNA carrying manipulated/interrupted gene whereafter homologous recombination occurs.
The DNA is then tested for resistance markers and inserted into the mouse blastocyst. The
first mouse made is chimeric. A chimeric mouse contains both normal cells and genetically
manipulated "knockout" cells. The chimeric mice breed to obtain heterozygous mice, which
can then breed for homozygous (normal) offspring).
RNA Interface (RNAi)
RNAi is used as approach to silence gene expression. It circumvents the problem of lethal
knockouts and uses short, non-coding RNAs with regulatory function. RNAi is a cellular
process/mechanism for post-transcriptional regulation. It silences genes which are not
required any more for development. It is also a defence mechanism against foreign DNA/
RNA.
In principle during RNAi, short double stranded RNAs are recognised and processed by a
specialised enzymes system known as Dicer. This form siRNA (small-interfering), which can
bind to mRNA. The mRNA is then cleaved by a specialised protein complex known as RISC
or Argo. This process leads to the inability of translation
Plasmids that code for siRNA are used a vehicle, which allows synthesis of siRNA and gene
silencing/knockdown.
RNAi can have off target effects and without the virus as vehicle, only short lived effects can
be produced. Also, a limitation is to produce a sufficient siRNA sequence
RNAi is applicated in ATP synthase inhibition. It can be used as drug target using BDQ-
type molecules as drugs. RNAi silences the ATP synthase gene expression by using
siRNA complementary with mRNA for the ATP synthase c subunit.
RNAi is also applicated to construct a plasmid for siRNA production via tetracyclic-
dependent transcription, which can transform into bacteria. Letting the bacteria grow,
RNAi production is activated with tetracycline. Is the bacteria then still viable? Results
show tetracyclic-dependent decrease of bacterial cell density
CRISPR-CAS
CRISPR/Cas is an adaptive immune system in bacteria and archaea Clustered
against viruses. The sequencing of E.coli reveals that is contains Regularly
short repeating sequences and it is interspaced with unique Interspaced
sequences, known as spaces. The system is accompanied by a Short
number of Cas genes which encode for Cas proteins, which are Palindromic
DNA cutting enzymes. The spacer sequences showed similarity to Repeats
viral sequences, and therefore it is investigated as defensive
mechanism. CRISPR offers resistance against viruses. The spacer CRISPR
sequences contain ASsociated genes
information on the
specific viruses.
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