lecture 1 - gene transfer to mammalian cells and gene therapy

Question 1

difference between forward and reverse genetics?

Answer 1

Forward genetics = start with phenotype and finding the associated gene
Reverse genetics = investigate the function of the genes

Question 2

what three tools do you need for studying the function of mammalian genes

Answer 2

1. Isolate and clone the gene of interest
2. Ability to manipulate the sequence of the isolated gene
3. Ability to return the altered gene back into eukaryotic cells to determine its function

Question 3

3 reasons for gene transfer to mammalian cells

Answer 3

1. research
2. biotechnology
3. gene therapy

Question 4

Isolate individual cell by…..

Answer 4

disrupting the extracellular matrix and cell junctions (cells are bound to extracellular matrix and cell junctions)

Question 5

why do most cell lines originate from tumors

Answer 5

tumor cells don’t have the normal limited cell growth
(Tumor cells have different properties from normal body cells -> development for normal body cell body cell lines is still going)

Question 6

why do mammalian cells require a solid surface?

Answer 6

after being released from the tissue environment, cells require a surface on which they can attach, otherwise they will fail to survive and divide
(Use polylysine/extracellular matrix components)

Question 7

how does a fluorescent-activated cell sorter work?

Answer 7

• Depended on having cells expressing on the outside labels that you can detect for example an antibody, which is marked with a fluorescent probe
• Droplets with the single cells are being analyzed whether the cell is fluorescent -> depending on fluorescence the cells get a negative or positive charge and are separated

Question 8

how does micro-dissection from tissue slices work

Answer 8

Take a tissue slice -> find the cells you want (vb cancer cells) -> laser beam cuts around region of interest -> second laser beam used to ‘catapult’ selected region into container

Question 9

primary cell cultures

Answer 9

cell cultures directly prepared from tissues of an organism

Question 10

immortalized cell line

Answer 10

a population of cells from a multicellular organism which would normally not proliferate indefinitely but, due to mutation, have evaded normal cellular senescence and instead can keep undergoing division

Question 11

how to keep isolated cells

Answer 11

• immortalize your cell line
• add growth factors to stimulate replication of specific cell types

Question 12

fibroplast

Answer 12

the cell type that predominates cultures of tissue or an embryo, they are the most common cells of connective tissue in animals

Question 13

methods for introducing DNA into mammalian cells

Answer 13

• calcium-phosphate co-precipitation
• electroporation
• lipofection
• viral vectors
• LTR retroviruses
• adenoviruses

Question 14

how does calcium-phosphate co-precipitation work?

Answer 14

• Cells take up DNA efficiently when it is in the form of a precipitate with calcium phosphate
• This method found that purified tumor virus DNA could transform normal cells into cancer cells

Question 15

how does electroporation work

Answer 15

• Cells are placed in a (salt-free) solution containing DNA and subjected to a brief electrical pulse that causes holes to open transiently their membranes
• DNA enters through the holes directly into the cytoplasm, bypassing the endocytotic vesicles through which they pass in the DEAE-dextran and calcium phosphate procedures
  
  • Passage through these vesicles
    may sometimes destroy or
    damage DNA

Question 16

what is lipofection

Answer 16

method to incorporate DNA into the cell, through plasmid-liposome complex-mediated gene transfer