Lecture 18, Chapter 11 Analysis of transgenic plants part I

Lecture 18, Chapter 11

Analysis of transgenic plantspart I

Mat Halter3/27/12

Plant Genetics, Breeding and Biotechnology (PLSC 452/552),University of Tennessee

Lecture 19, Chapter 11Analysis of transgenic plants

part II

Neal Stewart

pCAMBIA220111773 bp

lacZ alpha

Gus first exon

Gus second exon

chloramphenical (R)

kanamycin (R)

pVS1 sta

pBR322 bom

Catalase intron

T-Border (right)

T-Border (left)

Histidine tag

Nos poly-A

CaMV35S polyA

CaMV 35S promoter

CaMV35S promoter

pVS1 rep

pBR322 ori

Bam H I (10982)

Bst EII (2050)

Bst XI (10718)

Eco R I (10961)

Hin d III (11012)

Kpn I (10977)

Pst I (11004)

Sac I (10971)

Sal I (10994)

Sma I (10979)

Xba I (10988)

Pml I (2037)

Bgl II (8)

Bgl II (9918)

Nco I (1)

Nco I (9903)

Nhe I (2014)

Nhe I (5458)

Xho I (9053)

Xho I (9931)

Sph I (2455)Sph I (9373)

Sph I (11010)

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

500 bp

750 bp

1000 bp

1500 bp-

+

Ladder Sample

PCR and False Positives

Genomic DNATransgenic plant produced from Agrobacterium-mediated transformation

• 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?

LB RB

DNA ProbeEcoRI Site

• 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 T-DNA.

Why is a single cut within the T-DNA necessary?

LB RB RBLB RB

EcoRI Site EcoRI Site

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.

http://www.ndpteachers.org/perit/Electrophoresis%20%5B2%5D.gif

Restriction digest and gel electrophoresis

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

RNA loading controls are necessary to ensure an equal amount of RNA is loaded in each well.

No digestion necessary… why is this?

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 RT-PCR) 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.youtube.com/watch?v=QVeVIM1yRMU

http://www.rt-pcr.com/ 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