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Ectopic Gene Expression in Mammalian Cells
Definition: ectopic
Etymology: Greek ektopos out of place, from ex‐ out + topos place
occurring in an abnormal position or in an unusual manner or form Serap Tokmak
Transcription of a gene required presence of regulatory sequences and involves protein‐DNA as well as protein‐protein interaction − In eukaryotes, RNA polymerase, and therefore the initiation of transcription, requires the presence of a core promoter sequence in the DNA
− Promoters are regions of DNA, which promote transcription and are found around ‐10 to ‐35 base pairs upstream from the start site of transcription
− RNA polymerase is able to bind to core promoters in the presence of various specific transcription factors.
General transcription factors − bring the RNA polymerase to the correct position on the promoter
− help separating the DNA strands
Specific transcription factors
− can selectively regulate a gene or a gene group
− bind also the promoter / enhancer regions − Other proteins known as activators and repressors, along with any associated coactivators or corepressors, are responsible for modulating transcription rate.
Gene expression Vectors
− Contain regulatory elements similar to endogenous genes − Promoter choice depends on the aim of the experiment
− Minimal promoters result in low level basal transcription
− Constitutive promoters can drive expression of the gene regardless of the cell type
− Cell‐specific promoters lead to the expression of gene in a definite cell type − Regulated promoters can be turned on and off to control the expression of the gene
Ectopic Gene Expression in Mammalian Cells
• Gene transfer using − plasmid DNA (Physical or chemical non‐viral transfer)
− Virus
• Depending on the need
− transient or stable gene expression
• Analysis of the ectopic gene expression
− selection of the cells expressing the „transgene“
− antibiotic selection
− fluorescent gene expression
− protein expression
− DNA analysis
Ectopic Gene Expression in Mammalian Cells
Methods:
Non‐viral
‐Calcium phosphate Transfection
‐ Lipofection
‐ Electroporation
‐ Microinjection
Viral
‐Retroviral Transfer
Alternative methods with combinations of viral and non-viral transfer
methods
Calcium phosphate Transfection
− relies on precipitates of plasmid DNA with calcium ions
− DNA precipitates enter the cell by endocytosis
− Temperature and pH of the solution are important for an efficient transfection
Protocol:
− Day ‐1: HEK 293T cells are plated 1 day prior to transfection so that they are 50‐80% confluent on the day of transfection − 5x106 Cells in 10cm dish
− Day 0: Two solutions are necessary
− Solution A: DNA containing solution supplemented with a CaCl2 − Solution B: HEPES‐Buffered Saline(2x)
−Two solutions are mixed 1:1
−1µl Chloroquine is added to the medium of HEK293T cells prior to the addition of transfection mix
− incubate overnight at 37oC and 5% CO2
− Day 1: Transfection medium is replaced with fresh culture medium and the cells are incubated till further analysis
Transfection of HEK293T Cells with a GFP-Plasmid
-
+ Laserlight (488 nm)
(Fluorescence microscopy)
Lipofection: A lipid‐based transfection method
− Reagents −are generally composed of synthetic cationic lipids
− assemble in liposomes or micelles with an overall positive charge at physiological pH −are able to form complexes (lipoplexes) with negatively charged nucleic acids through electrostatics interactions
Electroporation
− the use of high‐voltage electric shocks to introduce DNA into cells − yields a high frequency of stable transformants and has a high efficiency of transient gene expression
− high‐voltage electric shock results in temporary breakdown of membranes and the formation of pores that are large enough to allow macromolecules (as well as smaller molecules such as ATP) to enter or leave the cell
Important criteria:
− Voltage (Volt)
− Impulse length
‐ Temperature
‐ DNA concentration
‐ Ionen combination in the electroporation medium
Protocol:
− Cells are placed in suspension in an appropriate electroporation buffer in a cuvette
− DNA is added and the cuvette is connected to a power supply
− The cells are subjected to a high‐voltage electrical pulse of defined magnitude and length
− The cells are then allowed to recover briefly before they are placed in normal growth medium
Microinjection
−injection of a DNA solution into the pronuclei of fertilized eggs is the most common method for making transgenic animals
− Materials to inject are
− DNA
− RNAi (Knockdown)
− Protein
− as well as the whole nucleus (to generate clones)
http://www.research.uci.edu/tmf/dnaMicro.htm
http://labspace.co.kr/bbs/data/products/eppen.jpg
Retroviral Transfer
Retroviruses:
− RNA viruses
− Retroviral upon infection of the target cell, the RNA genome is reverse
transcribed into DNA
− viral provirus integrates into the target cell genome
Vectors for Gene Transfer
virus
The Replication Cycle of Retroviruses
ctor
viral ve
translation:
protein of desire
(e.g. GFP)
Stucture of a Retrovirus
(RNA, 2x)
LTRs: long terminal repeats
Ψ+ (psi): packaging signal
Buchschacher, G. L. and Wong‐Staal F. 2000
Generation of retroviral particle
Wild type virus
LTR
Y
Transfer construct
(with therapeutic gene)
LTR
Y
Packaging plasmid
Envelope plasmid
gag
pol
env
LTR
Gen X
Y
gag
Y
env
LTR
pol
Y
LTR
Gen X
LTR
Y
gag
pol
Y
env
Transfection into a packaging cell
Packaging cell
Production and release of viral particles into the culture medium
Transduction of target cells
Selection/detection for transfected/transduced cells 1. Selection marker: Antibiotic resistence (i.e. puromycin)
transfection
selection
(1‐2 weeks)
experiment
2. Fluorescent protein: GFP, YFP, ....
transduction after 3‐4 days: experiment
Reporter genes must
− not interfere with the cellular functions in the target cell
− encode proteins that can be distinguished from the ones present in the target cell
− code for proteins that are readily detectable
Green Fluorescent Protein (GFP)
GFP mice
GFP lymphoma cells
Detection of transduced cells
via FACS: Fluorescence activated cell sorting
A flow cytometer has 5 main components:
• a flow cell ‐ liquid stream (sheath fluid), which carries and aligns the cells so that they pass single file through the light beam for sensing •an optical system ‐ commonly used are lamps (mercury, xenon); high‐power water‐
cooled lasers (argon, krypton, dye laser); low‐power air‐cooled lasers (argon (488 nm), red‐HeNe (633 nm), green‐HeNe, HeCd (UV)); diode lasers (blue, green, red, violet) resulting in light signals •a detector and Analogue‐to‐Digital Conversion (ADC) system ‐ which generates FSC and SSC as well as fluorescence signals from light into electrical signals that can be processed by a computer •an amplification system ‐ linear or logarithmic
•a computer for analysis of the signals. FSC correlates with the cell volume and SSC depends on the inner complexity of the particle (i.e., shape of the nucleus, the amount and type of cytoplasmic granules or the membrane roughness). Flow cytometry can be used to measure
•DNA (cell cycle analysis, cell kinetics, proliferation, etc.) •chromosome analysis and sorting (library construction, chromosome paint) •protein expression and localization •Protein modifications, phospho‐proteins •transgenic products in vivo, particularly the Green fluorescent protein or related fluorescent * cell surface antigens (Cluster of differentiation (CD) markers) •intracellular antigens (various cytokines, secondary mediators, etc.) •enzymatic activity •apoptosis (quantification, measurement of DNA degradation, mitochondrial membrane potential, permeability changes, caspase activity) •cell viability
•oxidative burst
Transduction of hematopoetic Leukaemic cell line
IRES
SFFV
RRE
eGFP
R
sa
CP day 1
CP day 10
96 %
102 103 104
95 %
100 101
102 103 104
100 101 102 103 104
NC128 day 1
100 101 102 103 104
NC128 day 10
11 %
102 103 104
96 %
100 101
sd
gag
100 101
Ψ
102 103 104
U5
100 101
R
CD34
U3
ΔU3
cPPT
Flag-NLS
Peptide
100 101 102 103 104
100 101 102 103 104
eGFP
U5
Insertional mutagenesis
vector
ATG tumor supressor
vector
oncogene
end
Gene Targeting
• transfer of a gene of interest into ES cells
• i. e. by electroporation
• selection of the transfected/ transduced cells • possible: PCR to identify transgene in the right locus
• transfer of engineered ES cells into pseudopregnant animal
Reverse Transcription of Retrovirus‐
1 Minus‐strand DNA synthesis is initiated using the 3′end of a partially unwound transfer RNA which is annealed to the
primer‐binding site (PBS) in genomic RNA, as a primer. Minus‐strand DNA synthesis proceeds until the 5′end of genomic RNA is reached, generating a DNA intermediate of discrete length termed minus‐strand strong‐stop DNA (–
sssDNA). Since the binding site for the tRNA primer is near the 5′ end of viral RNA, –sssDNA is relatively short, on the order of 100–150 bases
2 Following RNase‐H‐mediated degradation of the RNA strand of the RNA:–sssDNA duplex, the first strand transfer causes –sssDNA to be annealed to the 3′end of a viral genomic RNA. This transfer is mediated by identical sequences known as the repeated (R) sequences, which are present at the 5′ and 3′ends of the RNA genome. The 3′end of –
sssDNA was copied from the R sequences at the 5′end of the viral genome and therefore contains sequences complementary to R. After the RNA template has been removed, –sssDNA can anneal to the R sequences at the 3′end of the RNA genome. The annealing reaction appears to be facilitated by the NC.
3 Once the –sssDNA has been transferred to the 3′R segment on viral RNA, minus‐strand DNA synthesis resumes, accompanied by RNase H digestion of the template strand. This degradation is not complete, however.
4 The RNA genome contains a short polypurine tract (PPT) that is relatively resistant to RNase H degradation. A defined RNA segment derived from the PPT primes plus‐strand DNA synthesis. Plus‐strand synthesis is halted after a portion of the primer tRNA is reverse‐transcribed, yielding a DNA called plus‐strand strong‐stop DNA (+sssDNA). Although all strains of retroviruses generate a defined plus‐strand primer from the PPT, some viruses generate additional plus‐
strand primers from the RNA genome.
5 RNase H removes the primer tRNA, exposing sequences in +sssDNA that are complementary to sequences at or near the 3′end of plus‐strand DNA.
6 Annealing of the complementary PBS segments in +sssDNA and minus‐strand DNA constitutes the second strand transfer.
7 Plus‐ and minus‐strand syntheses are then completed, with the plus and minus strands of DNA each serving as a template for the other strand.
Aufbau [Bearbeiten]
LTRs enthalten alle Signalsequenzen, die zur Steuerung der Genexpression notwendig sind von 5' nach 3':
Abschnitt U3 (unique 3') mit GRE (charakteristische Basensequenz TGTTA), Enhancer
(TGTGCTAAG) und Promotor (TATA‐Box) Abschnitt R (redundant) mit einem Polyadenylierungssignal (AATAAA) für die Bildung eines poly(A)‐Schwanzes zur Stabilisierung der mRNA (siehe Transkription und Gen) Abschnitt U5 (unique 5') Funktionen [Bearbeiten]
LTRs können die Transkription initiieren, verstärken und steuern. Sie bieten Bindungsstellen für Transkriptionsfaktoren, die für die Gewebespezifität verantwortlich sind. Sie können aber auch die Transkription terminieren.