Aplikovaná chemie a biochemie
Přednáška č. 4
Manipulace genové
exprese
Modulace exprese nebo funkce proteinModulace exprese nebo funkce proteinůů::
•• chemickchemickáá inhibice, aktivaceinhibice, aktivace
•• zmzměěna genovna genovéé expreseexprese
•• selekce rezistentnselekce rezistentníích klonch klonůů
•• poupoužžititíí ppřřirozených mutantirozených mutantůů
•• overexpreseoverexprese proteinuproteinu
•• overexpreseoverexprese dominantndominantněě negativnnegativníích mutantch mutantůů
-- tranzientntranzientníí vsvs. stabiln. stabilníí transfekcetransfekce
•• poupoužžititíí antisenseantisense oligonukleotidoligonukleotidůů
•• RNA interferenceRNA interference -- tranzientntranzientníí a stabilna stabilníí
ChemickChemickáá inhibice,inhibice, aktivace:aktivace:
H-89
Forskolin
Různá specifita inhibitorů:
DalDalšíší kritickkritickéé body pro poubody pro použžititíí inhibitorinhibitorůů::
• rozpustnost;
• stabilita;
• biodostupnost;
• nežádoucí reakce s receptory;
• různá aktivita vůči izolovaným nebo rekombinantním
proteinům a v buněčné kultuře nebo v in vivo
podmínkách.
•• selekce rezistentnselekce rezistentníích klonch klonůů
•• poupoužžititíí ppřřirozenýchirozených mutantmutantůů nebo lininebo liniíí KO myKO myšíší;;
BuBuňňky jsou dlouhodobky jsou dlouhodoběě ppěěstovstováány v pny v přříítomnostitomnosti
úúččinných koncentracinných koncentracíí vysoce toxických lvysoce toxických láátektek -- napnapřř..
cytostatikcytostatik -- jsoujsou vyselektovvyselektováányny ppřřeežžíívajvajííccíí bubuňňkyky
schopnschopnéé rrůůst v pst v přříítomnosti toxických ltomnosti toxických láátektek -- napnapřř..
cytostatika, toxiny. Zpcytostatika, toxiny. Zpěětntněě jsou pak studovjsou pak studováányny
zmzměěny nany na úúrovni exprese proteinrovni exprese proteinůů..
ToxickToxickéé úúččinky cytostatika na ninky cytostatika na náádorovdorovéé
bubuňňkyky
VyuVyužžititíí bunbuněěk izolovaných zk izolovaných z knockknock--outout mymyšíší::
MEFs (mouse embryonic fibroblasts)MEFs (mouse embryonic fibroblasts)
Manipulace s funkcí proteinu prostřednictvím overexprese
identického genu se změněnou funkcí:
V podmínkách in vitro můžeme vhodný model získat buď z existujícího
organismu, nebo připravit gen kódující změněnou funkci uměle.
Transformace kompetentních
bakterií (E.coli)
- tepelný šok
- elektroporace
Izolace plazmidu
Transfekce bunTransfekce buněčěčných lininých liniíí in vitroin vitro::
TransfectionTransfection is the process of introducing naked DNA molecules into cells.is the process of introducing naked DNA molecules into cells.
Transfection can be categorized into 2 major types,Transfection can be categorized into 2 major types, stablestable andand transienttransient..
Transient transfection is temporary and high level expression ofTransient transfection is temporary and high level expression of foreignforeign
genes. Expression lasts for several days, but is lost as the DNAgenes. Expression lasts for several days, but is lost as the DNA nevernever
integratesintegrates stably into the host cell DNA. In contrast, stable transfectionstably into the host cell DNA. In contrast, stable transfection
occurs with a lower frequency (10 to 100occurs with a lower frequency (10 to 100--fold lower), but expression isfold lower), but expression is
maintained for the long term because the foreign DNA does integrmaintained for the long term because the foreign DNA does integrate intoate into
the host DNA.In the case of stable transfection, cotransfectionthe host DNA.In the case of stable transfection, cotransfection is oftenis often
used to introduce aused to introduce a selectable markerselectable marker (such as an antibiotic resistance(such as an antibiotic resistance
gene). Since only one in 1000 cells might be stably transfected,gene). Since only one in 1000 cells might be stably transfected, it isit is
necessary to select these cells fromthe total population. Cellsnecessary to select these cells fromthe total population. Cells that expressthat express
the selectable marker also take up and express the other gene ofthe selectable marker also take up and express the other gene of interest.interest.
LipofectionLipofection is a procedure in which the DNA is complexed within lipidis a procedure in which the DNA is complexed within lipid
droplets. The droplets interact directly with the cell membranedroplets. The droplets interact directly with the cell membrane and fuse.and fuse.
The DNA is liberated into the cytoplasm and some eventually reacThe DNA is liberated into the cytoplasm and some eventually reaches thehes the
nucleus. Lipofection is one of the most efficient methods of tranucleus. Lipofection is one of the most efficient methods of transfection.nsfection.
However, it is also relatively expensive, it can be toxic to celHowever, it is also relatively expensive, it can be toxic to cells, and itls, and it
cannot be used with cells growing in serum. Lipofection, like otcannot be used with cells growing in serum. Lipofection, like other forms ofher forms of
transfection, works much more efficiently if the cells are rapidtransfection, works much more efficiently if the cells are rapidly growing.ly growing.
This is because the nuclear membrane is absent during cell divisThis is because the nuclear membrane is absent during cell division, allowingion, allowing
easier access to the host DNA.easier access to the host DNA.
Calcium phosphateCalcium phosphate is another popular method for transfection. In thisis another popular method for transfection. In this
procedure, the DNA is precipitated with calcium phosphate aggregprocedure, the DNA is precipitated with calcium phosphate aggregates.ates.
The cells phagocytize the aggregates and the DNA is released intThe cells phagocytize the aggregates and the DNA is released into theo the
cytoplasm and eventually reaches the nucleus. This is the oldestcytoplasm and eventually reaches the nucleus. This is the oldest methodmethod
and its main advantage is that it is cheap and easy to perform.and its main advantage is that it is cheap and easy to perform.
However, calcium phosphate transfection is not as efficient asHowever, calcium phosphate transfection is not as efficient as
lipofection and the precipitates often causelipofection and the precipitates often cause cytotoxicitycytotoxicity..
ElectroporationElectroporation is another method of transfection in which cells areis another method of transfection in which cells are
exposed to an electric shock. This induces transient aqueous chaexposed to an electric shock. This induces transient aqueous channels innnels in
the membrane for DNA to enter the cytoplasm. On the positive sidthe membrane for DNA to enter the cytoplasm. On the positive side,e,
electroporation is rapid and simple, and it works on almost allelectroporation is rapid and simple, and it works on almost all types oftypes of
cells. However, one needs special equipment such as an electropocells. However, one needs special equipment such as an electroporator torator to
shock the cells. The transfected cells also have high cytotoxicishock the cells. The transfected cells also have high cytotoxicity afterty after
shocking.shocking.
There are severalThere are several viral vector systemsviral vector systems that have been developed forthat have been developed for
the study of gene expression in vitro or in vivothe study of gene expression in vitro or in vivo, including, including recombinantrecombinant
vaccinia viruses, retroviruses, and adenoviruses.vaccinia viruses, retroviruses, and adenoviruses. DDue to bioue to bio--safetysafety
regulations, a special lab facility must be available.regulations, a special lab facility must be available.
Metody selekceMetody selekce
ProkaryotaProkaryota
EukaryotaEukaryota
Overexpression/ectopic expression:Overexpression/ectopic expression:
• exprese velmi vysokých hladin proteinu v buňce, která
ho za normálních okolností neexprimuje, nebo jen v
omezené množství;
• nevýhodou je častá nespecifita – aberantní lokalizace
proteinu, aberantní interakce s dalšími proteiny, atd.
InducibilnInducibilníí exprese:exprese:
Doxycyclin - + +
Time (h) 0 24 48
- + - Doxycyclin (48 h)
- - + TCDD (48 h)
Cyclin A
ERK2
DNAsynthesis
(inductionx-fold)
0
1.0
2.0
3.0
4.0
DominantnDominantněě--negativnnegativníí mutantmutant –– definice:definice:
-- mutantnmutantníí protein, který potlaprotein, který potlaččuje funkci wt proteinuuje funkci wt proteinu
v pv přříípadpaděě spolespoleččnnéé exprese;exprese;
-- mechanismymechanismy –– multimerizace, titrace (upstream ormultimerizace, titrace (upstream or
downstream targets), aktivndownstream targets), aktivníí represe.represe.
KonstitutivnKonstitutivněě--aktivnaktivníí mutantmutant
AntisenseAntisense technology:technology:
Design a pDesign a přříípravaprava antisense oligonukleotidantisense oligonukleotidůů::
1) Chemistry - unmodified
phosphodiester DNA is rapidly metabolized
inboth serum and cells - several chemical
modifications can be incorporated into antisense
molecules to boost their nuclease resistance. Two
examples are phosphorothioate
oligodeoxynucleotides and 29-O-methyl
oligonucleotides.
2) Length - most antisense molecules
are 15–20 bases long, a length theoretically
sufficient to pick out a unique sequence from
others in the human genome and identify a target
mRNA (Ref. 18). Antisense oligomers of this size
have been successfully used to discriminate
between two gene products that differ by a
mutation of a single bas. Longer oligonucleotide
sequences (e.g. 30 nucleotides) aremore
expensive to synthesize and they might actually
increase the risk of non-sequence specific mRNA
cleavage because of growing probabilities that
other mRNA hybridization sites will be included in
long oligomers. Shorter oligomers, meanwhile,
generally do not have sufficient affinity to result
in adequate potency.
3) Sequence selection - empirical - not
all areas of a mRNA molecule are equally
amenable to antisense hybridization. The reasons
for this are unclear but probably involve mRNA
secondary structure, proteins bound to the mRNA
or accessibility of hybridized mRNA to RNase H.
‘gene-walk’ approach involves synthesizing
oligonucleotides that target regions scattered
throughout the entire mRNA sequence and then
evaluating these compounds in cell-culture assays
for antisense activity.
RNA Interference
RNA molecules have been used for
over two decades to reduce or
interfere with expression of
targeted genes in a variety of
systems. Historically, these
methods have been called post
transcription gene silencing (PTGS)
in plants, quelling in fungi and RNA
interference (RNAi) in higher
animals. Although originally thought
to require use of long doublestranded
(DS) RNA molecules, the
active mediators are now known to
be short DS RNAs. These short
interfering RNAs (siRNAs) are
naturally produced in vivo through
nucleolytic processing of long DS
RNAs. Short DS RNAs can also be
chemically synthesized and used to
experimentally inhibit gene
expression.
The Nobel Prize in Physiology or
Medicine 2006
"for their discovery of RNA interference gene
silencing by double-stranded RNA"
Andrew Fire Craig Mello
KritickKritickéé body pro aplikaci siRNA:body pro aplikaci siRNA:
• vhodně navržená sekvence (www.dharmacon.com; www.ambion.com);
• stabilita siRNA;
• vhodný způsob transfekce;
• optimální stav buněčné kultury;
• především optimálně nastavený systém kontrol a
vyloučení tzv. nespecifické interferonové reakce.
Praktické využití syntetické siRNA:
RNA Interference pomocRNA Interference pomocíí shRNAshRNA