VirusPhage Vectors:

1

• Virus/Phage Vectors
• -Overcome the size limits, can specialize the
  vector -SS DNA cloning -Expression cloning/gene
  therapy
• -higher efficiency (infection vs. transformation)
• Phage Lambda. A coliphage
• -Advantages Large inserts (50 kb!)-Headfull
  packaging Central segments of genome
  non-essential (outside arms or COS sites are
  all that required)-These arms ligated to RE cut
  donor DNA, chimeras are packaged into phage heads
  in vitro. -These phage are infectious. -The
  presence of a PFU automatically signals presence
  of inserted donor DNA
• Packaged Phage very stable, can be stored years
  for future screens.
• OVERVIEW ON NEXT SLIDE FIG. 12-7 TEXT

2
BamH1 GGATCCCCTAGG Sau3a GATCCTAG
Read over SS phage systems M13 (ss DNA
packaged into phage ). Useful for
oligonucleotide site directed mutagenesis and for
DNA sequencing (Sanger method)
3
Cosmid Cloning
Fig 12-8
-Hybrids of lambda and plasmids.-Replicate like
plasmid in cell, pack and deliver like the phage.
-Cos site cutting system packages phage
DNA-Requirements for packing 2 cos sites
separated by no less than 38 and no more than 54
kb.
4
Expression Vectors
To over-express a gene product, assist in a gene
hunt (by using antibodies to detect the presence
of a foreign gene product) one can use expression
vectors. Some considerations -no introns
allowed often a cDNA is used -Vector has
appropriate txn/tln start signals (lac system
works)
5

• A DNA Library a collection of clones
  representing a larger genome or sequence array
  set.
• -Shotgun cloning Random inserts of entire gene
  set or genome or whatever!
• -Could be a collection of phage (lambda) or
  bacteria (plasmids or cosmids, BACs) or even
  yeast (YACs)
• -Different libraries
• Genomic library represents all genomic DNA
• cDNA library copy DNA from mRNA (FIG- 12-9)
  Reverse transcriptase RNA dependent DNA
  polymerase (Rtase) reverse transcribes RNA to
  make a DS DNA copy
• Thus a cDNA library has no introns, regulatory
  elements 5 or 3.Ideal for expression based
  cloning but not so good for promoter analyses or
  getting at intron/exon organisation.
• BUT cDNA cloning is very useful for cloning
  overexpressed mRNAs (globin in reticulocytes)

6
Shotgun library Large member library, gene
hunts more difficult -To increase chances start
with a genetic fraction where sequence is
pre-selected EXAMPLE PFGE (oscillating field)
isolate whole chromosomes by gel electro.Or flow
cytometry (selection separation based on
size)-when you know which chromosome has YFG
7
Fig. 12-11

• Gene hunting Finding YFG
• Probe based DNA hybridization (simple rules of
  C)A. PROBES from many sources -cDNA in
  overexpressing tissue, like globin or ovalbumin.
  Can use HOT mRNA as well-bioinformatics on
  related organisms (conserved genes) or clone by
  phone-Reverse TLN aa with minimal redundancy
  reversed into DNA sequence (genetic code table)

Can make a cocktail probe or a degenerate probe
to screen library
8
Denatured DNA sticks to nitrocellulose filter
Genomic Libary
Gives a replica of pfu on plate. Hybridize with
a 32P labeled DNA or RNA or oligo probe
Use witness marks to re-orient the filter on
the bacterial lawn showing plaques.
Fig. 12-10
9
Gene hunting Finding YFG (continued) ANTIBODY
PROBESA. You have a purified protein inject
into mouse, rabbit, goat, OSU undergraduates,
etc. and make a monospecific ANTIBODY probe. B.
Screen an expression based library to find clone
expressing this ptn.
Fig. 12-12
10

• An example Get the gene for albinism
• Mechanism based we know defect is in an enzyme
  (tyrosinase)
• Purify enzyme, inject rabbit, pull serum (use as
  is or purify back IgG)
• From mRNA cells known to synthesize tyrosinase,
  make a cDNA expression based library (lambda)
• cDNA clones identified by an AB screen with
  above probe
• cDNA clone grown sequenced and shown to be 1590
  bp long (only Exons)
• cDNA now used to back hybridize/screen a human
  genomic DNA library
• Genomic clone isolated, sequenced
• Exon/intron arrangements worked out
• Structure/function studies of tyrosinase carried
  out (Mutagenesis of gene)
• FUTURE carry out gene therapy expts to
  correct defect in somatic cells.
• Not feasible as yet.

Coding
5 reg. elements
3 stuff
Delivery vehicle replication proficient,
integrative?, harmless to host, tissue
specificitymany others?