Transcript Plant Genetics, Breeding and Biotechnology (PLSC 452/552)

Lecture 18, Chapter 11
Analysis of transgenic plants
part I
Mat Halter
3/27/12
Plant Genetics, Breeding and Biotechnology (PLSC
452/552),
University of Tennessee
Lecture 19, Chapter 11
Analysis of transgenic plants
part II
Neal Stewart
Hin d III (11012)
Sph I (11010)
Pst I (11004)
Sal I (10994)
Xba I (10988)
Bam H I (10982)
Sma I (10979)
Kpn I (10977)
Sac I (10971)
Eco R I (10961)
Bst XI (10718)
Ca M V 3 5 S prom ote r
Xho I (9931)
Bgl II (9918)
Nco I (9903)
k a na m yc in (R)
Sph I (9373)
Xho I (9053)
Ca M V 3 5 S polyA
T-Borde r (le ft)
la c Z a lpha
Ca M V 3 5 S prom ote r
G us firs t e x on
Nco I (1)
Bgl II (8) Ca ta la s e intron
G us s e c ond e x on
His tidine ta g
Nhe I (2014)
Pml I (2037)
Bst EII (2050) Nos poly-A
T-Borde r (right)
Sph I (2455)
pCAMBIA2201
11773 bp
c hlora m phe nic a l (R)
pBR3 2 2 ori
pBR3 2 2 bom
pV S 1 s ta
Nhe I (5458)
pV S 1 re p
Transformation is a relatively rare event.
• Therefore selection has been needed.
– NPTII
– Bar
• Recently, easily scorable and non-invasive
markers.
Figure 9.3
Sometimes “escapes” occur– for kanamycin
resistance markers tissue is red—very stressed
Stable integration of transgene
• Transgene is permanently integrated into the
genome of the host plant.
• Transmitted to progeny (Tn plants) in
Mendelian fashion
• Need convincing proof of stable integration
• Multiple assays are possible—but most
researchers are best convinced by Southern
blot data.
Fluorescent Proteins
http://en.wikipedia.org/wiki/File:FPbeachTsien.jpg
PCR analysis by gel electrophoresis
-
Ladder
1500 bp
1000 bp
750 bp
500 bp
+
Sample
PCR and False Positives
Transgenic plant produced from
Agrobacterium-mediated
transformation
Genomic DNA
• In T0 plants, Agrobacterium left over from the initial
transformation is still present in all tissues.
• Contamination of the genomic DNA with the initial transformation
vector that is still present in the agrobacterium can produce a PCR
band.
Southern Blot
• Southern blotting confirms the presence of
the gene of interest in the genomic DNA of the
target plant and avoids the pitfalls of potential
false positives.
• Steps
– Genomic DNA isolation
– Restriction enzyme digestion of genomic DNA
– Running digested DNA on agarose gel to separate
fragmented DNA by size.
– Transfer of separated DNA to nylon membrane
– Hybridization with radioactive DNA probe
Digested Genomic
• Essentially, every known
restriction enzyme will have cut
sites in a plant genome.
• How can enzyme selection be
used to detect copies of an
inserted transgene?
EcoRI Site
DNA Probe
LB
RB
• Single cutting enzymes can be
designed into the T-DNA before
transformation that will enable
proper digestion of the genome as
well as a single cut within the TDNA.
Why is a single cut within the T-DNA
necessary?
EcoRI Site
EcoRI Site
LB
RB
LB
RB
If there is no EcoRI site within the tDNA, after digestion with
EcoRI these two insertion sites will be indistinguishable from
one another after electrophoresis and probing.
Cutting within the T-DNA is necessary to distinguish each
and every insertion event. This is VERY important.
Restriction digest and gel electrophoresis
http://www.ndpteachers.org/perit/Electrophoresis%20%5B2%5D.gif
Southern blot—DNA transfer to nylon
www.gbiosciences.com/Southern-Blot-desc.aspx
Northern blot analysis
• Gives relative amount of gene expression-at the
transcript level.
• Isolate mRNA be a lot and of good quality (not
degraded)
• Separate transcripts on a gel
• Transfer to nylon filter
• Probe filter with DNA of interest (transgene)
http://www.youtube.com/watch?v=KfHZFyADnNg
Northern Blot
No digestion
necessary… why is
this?
RNA loading controls
are necessary to
ensure an equal
amount of RNA is
loaded in each well.
Figure 11.9
Northern blot example
What is missing in this experiment?
Western blot
• Also to measure gene expression—at the
protein level.
• Extract proteins
• Separate proteins on a vertical gel
• Transfer to a membrane using an
electrotransfer system
• Probe with antibodies.
• Stain for antibodies
Western blots and ELISAs often use
amplification of signal via antibodies
http://probes.invitrogen.com/handbook/images/g001474.gif
Figure 11.11
Western blot example
What is missing in this experiment?
Real-time PCR or Quantitative PCR
• Real-time PCR uses fluorescence as an output
for DNA amplification in real-time.
• The amount of starting template DNA (or
cDNA for RNA measurement (real-time RTPCR) is correlated with the Ct number.
• More DNA = lower Ct; Ct is the cycle number
when a threshold amount of DNA is produced
during the PCR experiment.
http://www.rt-pcr.com/
http://www.youtube.com/watch?v=QVeVIM1yRMU
Advantages of qRT-PCR
over RT-PCR?
Summary
• Is my plant transgenic?
– Survives selection
– Reporter gene
expression
– Progeny analysis
– PCR
– Southern blot analysis
• Is my plant expressing
the transgene?
–
–
–
–
–
Northern blot analysis
Western blot analysis
ELISA
RT-PCR
Real-time RT PCR