1. Transfection of Mammalian Cells MBIOS 520/420 October 6, 2005

2. Gene Manipulation There are three steps to study the function of gene: I. Identify and clone the gene. II. Alter the gene to study its function. (create a knockout or specific mutation) III. Re-Introduce the altered gene back into the organism and analyze the phenotype. We’ve already covered ways to achieve Step I How do we accomplish Step II & III?

3. Altering a Gene There are several ways we can alter a gene We can insert a transposon or marker gene to knock it out Or we can make specific point mutations that will change the codons (and thus the amino acids) of the gene/protein: This is called site-directed mutagenesis (your book calls this oligonucleotide directed mutagenesis

4. Site-Directed Mutagenesis Let’s say we want to change a single codon in exon 2 of Gene X from AAT to ACC. We have Gene X in a plasmid, so we create a primer identical to part of exon 2, but with the CC pair we want instead of AT. We anneal this to Gene X and do PCR around the plasmid (similar to inverse PCR). The copies that are made have incorporated the mutations in the primer, changing the codon to ACC. AAT Plasmid w/ Gene X ACC Primer w/ changed codon Denature & anneal mutant primer PCR w/ mutant primer Product after 1 st cycle Mutant product after many cycles

5. Mammalian Transfection Techniques I. DEAE Dextran II. Calcium-Phosphate Co-Precipitation III. Electroporation IV. Microinjection V. Liposome-Mediated Uptake VI. Viral Vectors

6. Mammalian Transfection Techniques I. DEAE Dextran II. Calcium-Phosphate Co-Precipitation III. Electroporation IV. Microinjection V. Liposome-Mediated Uptake VI. Viral Vectors

7. Transfection Once we’ve created a mutant, we need to complete Step III and introduce it into a cell Mammalian cells have defenses against accepting foreign DNA, designed to protect against viruses Early methods involved coupling DNA to positively charged DEAE-dextran, which “stuck” to cells and entered via endocytosis Most of the DNA was destroyed by the cells’ defenses, so the method was inefficient However, when DNA is precipitated with calcium phosphate, cells take it in much more efficiently

8. Transfection by Ca-phosphate Co-precipitation In early experiments, researchers precipitated viral DNA & added it to mammalian cell culture 100X more viral particles were produced than when DEAE-dextran was used Because these early experiments used viruses, the process was called transfection: (transformation-infection) Efficiency is still 1/1000 cells

9. Transfection Just like in yeast, we need selectable markers to detect which cells took up DNA For mammals, one of the earliest selectable markers used was thymidine kinase (tk), since tk- cells had been isolated tk allows cells to “salvage” free pyrimidines and convert them into thymine or adenine nucleotides a different enzyme, HPRT, can salvage purines aminopterin is a drug that blocks nucleotide synthesis; thus cells with aminopterin must rely on tk & HPRT activity Basis of selection is co-transfection: the phenomenon that cells transfected with two DNA fragments by Ca precipitation will usually take up both

10. Co-Transfection & tk selection In this experiment, tk gene and a globin gene are precipitated via Ca- phosphate & introduced into mammalian tk - cells. Cells are grown on HAT medium to select for tk +. HAT MEDIUM H = hypoxanthine (allows HPRT to produce purines) A = aminopterin (blocks nucleotide synthesis) T = thymidine (allows tk to produce pyrimidines) When cells are isolated & Southern blots performed, cells have integrated both globin & tk genes into their genomes. NOTE: We could do the same thing for HPRT- cells.

11. High Protein Expression Through Gene Amplification Very few cells will actually integrate new DNA into their genome, but many will transiently express introduced DNA neo r, a resistance gene to neomycin drug G418 is the most commonly used selectable marker When researchers want to study a protein (to determine its structure or make antibodies), they need it in large quantities DHFR (dihydrofolate reductase) is a critical metabolic enzyme for creating nucleotides and is inhibited by methotrexate (Mtx) Some cells can survive methotrexate treatment by amplifying the DHFR gene through replication/recombination Researchers can attach a gene of interest to DHFR and it will be amplified along with it, making many copies & lots of protein

12. Gene Amplification Put both Gene X and DHFR in vector with a strong promoter (ex: cytomegalovirus or CMV) Mtx DHFR Transfect DHFR- cells with the vector & grow on nucleoside-free medium. Apply increasing concentrations of Mtx. Only a few cells will survive. Those that survive will have large numbers of vector DNA amplified Grow these cells & isolate large amount of Protein X.

13. Mammalian Transfection Techniques I. DEAE Dextran II. Calcium-Phosphate Co-Precipitation III. Electroporation IV. Microinjection V. Liposome-Mediated Uptake VI. Viral Vectors

14. Electroporation Calcium phosphate co- precipitation does not work in every cell type (ex: lymphocytes) Electroporation uses an electrical pulse that punches holes in the plasma membranes of cells so DNA can enter This method is very efficient, but usually kills > 50% of the cells because it is damaging

15. Mammalian Transfection Techniques I. DEAE Dextran II. Calcium-Phosphate Co-Precipitation III. Electroporation IV. Microinjection V. Liposome-Mediated Uptake VI. Viral Vectors

16. Microinjection of DNA Uses a computer-controlled needle to inject DNA directly into the nucleus of a cell Very reliable, but only be performed on one cell at a time

17. Mammalian Transfection Techniques I. DEAE Dextran II. Calcium-Phosphate Co-Precipitation III. Electroporation IV. Microinjection V. Liposome-Mediated Uptake VI. Viral Vectors

18. Liposome-Mediated Gene Transfer Artificial lipid vesicles (liposomes) can be created by forming a bilayer around DNA These capsules adhere to the cell membrane and fuse into it Making liposomes is complicated, but available commercially

19. Mammalian Transfection Techniques I. DEAE Dextran II. Calcium-Phosphate Co-Precipitation III. Electroporation IV. Microinjection V. Liposome-Mediated Uptake VI. Viral Vectors

20. Viral Vectors for Gene Transfer Viruses have the natural ability to successfully introduce DNA into foreign cells Normal plasmid vectors are modified by adding the viral genome, with gene of interest replacing the viral late genes Without the late genes, these viruses cannot replicate, so a helper virus (lacking early genes) is co-transformed with the plasmid Transformed cells produce both viruses carrying gene of interest Virus is isolated & transformed into new cells, which are unable to produce viruses (late genes missing) but produce the gene of interest

21. SV40 Viral Vector Use In this experiment, tk gene and a globin gene are precipitated via Ca- phosphate & introduced into mammalian tk - cells.

22. Bacculavirus Vector Use In this experiment, tk gene and a globin gene are precipitated via Ca- phosphate & introduced into mammalian tk - cells.

23. Mammalian Gene Knockouts In this experiment, tk gene and a globin gene are precipitated via Ca- phosphate & introduced into mammalian tk - cells.

24. Using PCR to Detect Gene Knockouts In this experiment, tk gene and a globin gene are precipitated via Ca-phosphate & introduced into mammalian tk - cells.