1 12. Capita selecta 1. SV40 vectors and transformation of

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12. Capita selecta

1. SV40 vectors and transformation of mammalian cells

Simian virus 40

(SV40)

Isolated from Rhesus monkey

Papovaviridae

Grows on kidney cells of monkey (AGMK, CV1, …)

Structure :

5243 bp, circular DNA chromatine-like structure (nucleosomes) virus particle : icosahedral, VP1, VP2, VP3

Genetical map : "

early

" & "

late

" region, O

R

Lytic development versus cellular transformation

Infection process uncoating : 8 h early phase: early mRNA's, early proteins

(large-T and small-t antigen)

: 4 u start of replication + late phase : late mRNA's and proteins : from +/- 36 u maturation : packaging, cell lysis

DNA replication : O

R

: ORI (core 80 bp) ; bidirectional ;

T-antigen required every time

Role of T-antigen : COS cells :

- integrated copy of SV40 DNA, with part of ORI deleted

- produces T-antigen constitutively

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Vectors :

- transducing vectors (with helper virus) : packageable, replicating

=> double infection is possible

=> early replacement and late replacement

- plasmid vectors : non-packageable, replicating

- passive vectors : non-inpackable, non-replicating (autonomously)

- the latter two types of vector usually bear part of pBR322 and also replicate in

E.coli

: hence, they are "shuttle vectors"

=> it was later found that the region around the

nic/bom

site of pBR322 causes poor transformation efficiencies in mammalian cells and better is removed. It is also called a "toxic region".

"Cloning” in mammalian cells

- principle of ‘co-transformation’ : during passage across the cytosol (towards

the nucleus, there is extensive ligation activity

-

transient

versus

permanent

(‘stable transformation’)

=> ‘amplification’ using methotrexate ( inhibitor of DHFR

) methotrexate is a competitive inhibitor of DHFR in general: resistance may develop by one of three mechanisms

- mutations in the

DHFR

gene

- inhibiting uptake of the inhibitor

- increased target dosage effect : amplifying gradually a region surrounding the gene amplification seems to be a more general response mechanism in higher cells

- markers and selection

- from the nucleotide metabolism (

TK

,

HPRT

,

APRT

, ...)

- exogenous, dominant markers (

neo

,

nptII

,

(

aphII

)

: geneticine; ...)

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- many reporter genes are used, often based on detection of color or fluorescence by enzymatic action : CAT, LacZ, GusA, luciferase, alkaline phosphatase.

=> a more recent addition to these is Green Fluorescent Protein (GFP) under UV (blue) light => green fluorescence (and variants)

=> advantage : does not require a substrate

2. Gene cloning in yeast (

Saccharomyces cerevisiae

)

Saccharomyces cerevisiae

: genome size: 12 Mb (excl. some repeats, a.o. rRNA); haploid set of chromosomes: 16 unicellular eukaryote

insight in eukaryotic processes

recombinant protein expression / possibilities of secretion and fermentation / no pyrogenic toxins, but glycosylations are possible, relatively high yields of pure proteins

easier to grow than animal cells

cloning of large DNA feasible (YAC vectors)

many auxotrophic and other genetical markers available

3

Genetic transformation

- no natural transformation procedure

-

(early methods)

use of

sphaeroplasts/protoplasts

(i.e. cells without cell wall)

– enzymatic treatment + permeabilisation with polyethylene glycol, treatment with CaCl2 + addition of DNA ; regeneration of the cell wall in medium containing 3% agar

- newer methods: use of Li-acetate, PEG, ssDNA (pretreatment) ; enzymatical removal of cell wall not required

- alternatives : electroporation, conjugation to

protoplastes

-

no useful yeast viruses => no transduction, no infection, no viral vectors

Vectors

- replicating circles or integration

- markers required : no antibiotic resistance, but G418 (geneticin) most of the markers based on auxotrophy : URA3, HIS3, etc negative and positive selection with URA3 : use of 5-FOA

Types of vectors : (see figures and tables)

YIp integration, recombination

YRp

YEp replication : ARS sequence episomal : derived from endogenous 2

plasmid

YCp ARS + centromere (CEN)

YAC artificial chromosome : ARS + CEN + telomeres

Homologous recombination is very efficient.

Directed exchanges by homologous recombination with linear fragments :

- the terminal sequences (> 30 bp) must be identical to the targeted ends

- the internal part can be entirely different from the natural sequence

- any target can be approached by PCR, with primers that bind with their

3’ terminal to the required amplicon, and have extension complementary to the yeast genomic target

Genome-wide analysis : ‘knock-out’ strain collection

- bar-coded deletion cassettes: every ORF as a target

- ‘ left side target

’- ’

20bp bar-code

’- ‘

Km resistance gene

’-’

20bp bar-code

’- ‘ right side target

- left & right side target are 40 bp each, identical to the sides of a targeted ORF

- the 20bp bar-codes are unique 20-mers, absent in the yeast genome, having about 50% G+C and similar Tm values

- the bar-codes are flanked by common PCR primer sequences so that they can be amplified simulaneouly (they are signatures of the particular targeted ORF, and readily identifyable on a DNA microarray containing the (approximately 12,000) bar-code sequences

- the Km-resistance gene (from bacterial origin) is flanked by a promotor and termination region active in yeast, with minimal sequence homology to other yeast genomic sequences ( to avoid secondary

(= false)

insertion sites

)

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- transformation into diploid yeast, upon selection with kanamycin, lead to heterozygous mutants, from which homozygous haploid deletants

(if viable) and, in turn, homozygous diploids

(if viable)

are derived for functional assays.

- a pool of many (or all) different mutants strains thus can be analysed in a single experiment by amplifying the bar-codes and probing the microarray.

(e.g. to follow the survival capacity under different stress conditions)

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