Transcript 投影片 1

Chapter 9
Introduction of DNA into Mammalian cells
Cells needs to be healthy before tansfection.
1. Check ATCC or BCRC catalog for culture methods ofcell
lines.
2. Tissue culture glassware should be soaking in water after
use, to prevent drying of residual chemicals into the
glass.
3. Heated inactivated (1 hr at 56oC) or non-heated fetal
bovine serum (also known as fetal calf serum, stored at
-20oC) can be added before use.
(Hyclone is a reliable supplier of fetal bovine serum).
4. Solutions are filter sterilized through Nalgene filters.
5. Do not use stir bars.
6. 2-ME (2-mercaptoethanol) is not required for culturing cell
lines but essential for culturing primary cultures.
Methods of transfection
－ Calcium phosphate method
1. Calcium phosphate and DNA form coprecipitates, which will attach to the surface
of the target cell.
2. Once attached, the DNA is endocytosized.
3. Up to 10% cell will take up the DNA.
4. Calcium phosphate aggregates DNA. The
method is good for co-transfection, or for
insertion of multiple copies in tandem array
into a single site.
－ liposome method
1. Negatively charged DNA (or RNA) binds to
positively charged surface of the liposome.
2. Residual positive charge of liposome
mediates binding to negatively charged sialic
acid residues on the cell surface.
3. Amounts of liposome, DNA, and the exposure
time are different with cell types, and may be
critical for transfection .
4. Liposome aggregates DNA. The method is
good for co-transfection, or for insertion of
multiple copies in tandem array into a single
site.
－ electroporation
1. for transfecting large DNA molecules.
2. can transfecting plant protoplasts.
3. introduces one copy at low concentrations of
infecting DNA.
4. At high DNA concentrations, about 25% of
cells have multiple copies inserted at a single
site.
－ viral vectors
1. Use baculovirus or vaccinia virus for over
expression of proteins.
2. Use retrovirus for stably introducing genes into
a cell line, primary cells, and cells in an animal.
- If multiple genes are desired, use separate
plasmids for selectable markers and genes.
- If a single gene is desired, use
electroporation. The selectable marker
and the gene are in a single plasmid.
- Transfection can be transient or stable.
- For obtain stable cell line, need a dominant
selectable marker.
-Transient expressions do not need selectable
marker.
In eukaryotic cells, a gene needs the following for transcription.
1.
2.
3.
4.
Promoter
Transcriptional start site
Polyadenylation site
Intervening sequence (intron) and splice site
Translation starts from the first AUG (GUG).
Translation efficiency from the second ATG is less than 10% of
that of the first AUG.
------------------------------------------------------------------------------------In prokaryotic cells, transcription need only promoter sequence
(-35 and -10, -24 and -12 or pribnow box sequence in E. coli).
Translation from an AUG with an ribosome binding sequence
(AGGA in E. coli).
Several genes can form an operon with polar effects.
1. Promoter: usually < 1kb, contain TATA box or
AGAC at +28 to +32.
Most genes contains enhances downstream (mostly)
or downstream of coding sequence for efficient transcription.
Frequently used constitutive promoters:
Promoter from CMV (cytomegalovirus)
Promoter from SV40 (simian virus 40),
Some with SV40ori for replication in cells
expressing T antigen.
pMC1
PGK (promoter from phosphoglycerate kinase)
Promoter from CaMV (cauliflower mosaic virus,
plant promoter)
S35 promoter (promoter from Rubisco, plant promoter)
2. Transcriptional start site:
Transcription is efficient with a Kozak
sequence, and follows Kozak rule.
3. Polyadenylation site:
Need sequence AAUAA, 11 to 30 nucleotides
upstream of the polyadenylation site, and a
GU- or U-rich region downstream.
4. Intervening sequence (intron) and
splice site:
Intron and mRNA splicing increase transcription.
Need 5’ and 3’ conserved splice sites.
After integration of single copy at a unique site, use Cre-lox system to
remove marker.
Amplification can be selected by gradually
increasing the selection pressure on the marker
gene (e,g., DHFR), by higher concentrations of
selection medium.
This results in tandem duplication of the
introducing gene.
To introduce a DNA at a specific site, use Cre-lox recombinase system. The
effects of different introduced DNAs can be compared at same location.
To obtain a stable transfected cell line,
1. Ck if the cell line can survive from 2% methylene blue
as single cells. If not, forget it.
2. Co-transfection by electroporation using 5:1 molar
ration of plasmid containing the gene to
plasmid containing the marker (linearization of plasmid
helps transfection).
3. Allow cells to double twice non-selectively, and split
1:15 into selective medium.
4. Feed cells with selective medium every 4 days or as
needed.
5. After 10-12 days, Ck plates for colonies. Pick good one
(about 500 to 100 cells) and culture, or select once and
culture.
Positive selection markers:
Adenosine deaminase (ADA)
Aminoglycoside phosphotransferase (neo, G418, APH)
Bleomycin (bleo, phleo, zeocin)
Cytosine deaminase (CDA, CD)
Dihydropholate reductase (DHFR)
Histidinol dehydrogenas(hisD)
Hygromycin-B-phosphotransferase (HPH)
Puromycine-N-acetyltransferase (PAC, puro)
Thymidine kinase (TK)
Xanthine-guanine phosphoribosyltransferase (XGPRT, gpt)
Negative Selection markers:
Cytosine deaminase (CDA, CD)
Diptheria toxin (DT)
Herpes simplex virus thymidine kinase (HSV- TK), ＋ to －
Used for selection of recombinants from homologous
recombination or the lox-cre system.
Reporter systems in eukaryotic cells
To be a transcriptional reporter gene,
1. The reporter protein should be a dead-end in cells.
2. The reporter protein should be absent or distinguishable
from the host.
3. Assay for reporter proteins needs to be simple and rapid.
4. (for quantitation) The assay should have broad linear
range.
5. Expression of the gene must not alter the physiology of
the recipient cells or organism.
- Gene expression can be assayed within 48 hrs after
transfection.
- Can find promoter, enhancer, or tans-acting elements.
In vitro reporter genes (use cell lysates or cultural medium)
CAT (chloramphenicol acetyl transferase)
CAT-ELISA with anti-CAT antibody (BM)
Fluorescenct chloramphenicol as substrate (Mol. Probe).
Firefly luciferase (luc) and Renilla luciferase (Rluc)
Dual reporter system from Promega.
β-Galactosidase
ONPG as substrate.
Secreted Alkaline phosphatase (SEAP)
Use culture medium for assay
Chemiluminescence assay (BD-Clontech)
β-Glucuronidase (Gus)
Good for protein localization and transport studies.
Used mostly in plants, because most plants lack
endogenousβ-glucuronidase.
In vivo reporter assays (live cells, fixed cells, and organisms)
Green fluorescent protein (GFP)
- vectors with GFP expression (for prokaryotic or for
eukaryotic, BD-Clontech)
- C- and N- terminal fusions are OK.
- Good with localization studies.
- Wt GFP Excited at 395nm, and 475nm; emit light at
509nm.
- Mutant GFPs with enhanced emission or different
excitation and emission spectrum are available
β-Galactosidase
X-gal as substrate.