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 56 o C) or non-heated fetal bovine serum (also known as fetal calf serum, stored at -20 o C) 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 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 co- precipitates, 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.Promoter 2.Transcriptional start site 3.Polyadenylation site 4.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 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.