This slide is about the characteristics of transgenic fish and how do we identify it.. e.g. by blotting, PCR, gene-X, etc.

1. By:
Saadia Laraib
Roll no. 30
6th Semester (A)

2. Steps for Making Transgenic
Fish
Step 1. Decide Gene/Protein to Add.
Step 2. Decode Protein and Translate to cDNA
Code.
cDNA
Protein

3. Steps for Making Transgenic
Fish
Step 3. Prepare Gene Construct.
Protein Gene Promoter Gene Gene Construct

4. Steps for Making Transgenic
Fish
Step 4. Insert Construct into Bacterial Plasmid.

5. Steps for Making Transgenic
Fish
Step 5. Insert Plasmid in
Bacterial Strain & Make
Billions of Copies.

6. Steps for Making Transgenic
Fish
Step 6. Isolate Plasmids from Bacteria
and Cleave into Linear Cassettes.

7. Steps for Making Transgenic
Fish
Step 7. Insert Over 1 Million Cassettes into
Each Newly Fertilized Egg.

8. Steps for Making Transgenic
Fish
Step 8. Incubate and Grow Out Surviving Fry.

9. Steps for Making Transgenic
Fish
Step 9. Find the Transgenics and Select
Fish(s) with Desired Characteristics.

10. Steps for Making Transgenic
Fish
Step 10. Breeding Program to Stabilize Transgene.

11. SOUTHERN BLOTTING
 1. Extract and purify DNA from cells
 2. DNA is restricted with enzymes
 3. Sort by electrophoresis
 4. Denature DNA
 5. Transfer to nitrocellulose paper
 6. Block with excess DNA
 7. Wash off unbound probe
 8. Autoradiograph

12. Looking for Gene X
extract
DNA
? copies of gene X

13. Step 1. Restriction Enzyme Digestion
EcoR I EcoR I EcoR I EcoR I

14. Step 1. Restriction Enzyme Digestion

15. Step 2. Gel Electrophoresis
_ +

16. Step 2. Gel Electrophoresis
_ +

17. Step 2. Gel Electrophoresis

18. Goals of Southern Hybridization
Immobilize DNA onto a permanent substrate
 ‘Membrane’
 paper-like matrix
 nylon or nitrocellulose
 usually has a slight positive charge

19. Step 3. DNA Denaturation
• Eliminate hydrogen bonds with sodium
hydroxide (NaOH)
A C T T G A
T G A A C T

20. Step 4. Transfer DNA to Membrane

21. Step 6. Pre-hybridization
Prehybridization buffers
contain ‘blocking reagents’
that occupy available binding
sites on the membrane

22. Step 7. Hybridization

23. Step 7. Hybridization

24. Step 8. Washes

25. Step 9. Anti-DIG

26. Step 9. Anti-DIG

27. Step 10. Washes

28. Step 11. CSPD

29. Step 12. Detection
 DIG-labeled probes
emitting minute
amounts of light
(chemiluminescence)
 32P-labeled probes
emitting ß-particles

30. Step 12. Detection
 DIG-labeled probes
emitting minute
amounts of light
(chemiluminescence)
 32P-labeled probes
emitting ß-particles
 Autoradiography film
can detect this
radiation

32. Results:
 How many copies of
‘Gene X’ does the fish
possess?
3

33. Dot Blot/ Northern Blotting

34.  extraction of total RNA
 separation by gel electrophoresis.
 Running through nylon membrane
 hybridized to the RNA on the membrane
to make cDNA
 washing
 The hybrid signals are then detected by
X-ray film

35. SCREENING BY PLAQUE /
COLONY HYBRIDIZATION
This method also utilizes a DNA probe. This is used
for colony hybridization.
The procedure involved is as follows
1. Transfer some of DNA in plaque / colony to a
nylon / nitrocellulose membrane. Because plaques
are areas of lysed bacteria, the phase DNA is
directly available & will bind to the membrane when
top of the petridish.
This can be achieved by soaking in sodium dodecyl
sulphate & protease.

36. 2. The DNA on the membrane is denatured with alkali to produce
single strands; deprotenised & is bonded to the membrane by
baking or UV radiation.
3. The membrane is then immersed in a solution containing a
nucleic acid probe, which is usually radioactive & incubated to
allow probe to hybridize to its complimentary sequence.
4. After hybridization, the membrane is washed extensively to
remove unhybridised probe.
5. The region where the probe has hybridized is visualized by
exposure to X-ray film.
6. A colony on the master plate that corresponds to the region
of a positive response on the X- ray film is identified. Cell from
the positive colony on the master plate are sub cloned because
the carry the desired DNA.
Thus the identification of gene of interest from the whole
genomic library is achieved.

38. Detection of Specific RNAs

39. Annealing of Downstream Primer to
RNA

40. Reverse Transcription With AMV
Reverse Transcriptase

41. RNA Copied From 3’ to 5’ into
cDNA

42. Amplification of cDNA by
PCR

44. This method is used when a DNA probe is not available.
In this method all the clones of the library are grown separately on a
plates. A sample of each colony is transferred to a known position
on a matrix, where the cells are lysed & the released proteins are
attached to the matrix.
The matrix with the bound proteins is treated with an antibody
(primary antibody) that specifically bounds to the protein encoded
by the target gene.
Following the interaction of primary antibody with the target protein
(antigen), any unbound antibody is washed away, and matrix is
treated with a second antibody (secondary antibody) that is specific
for primary antibody.
In many assay systems, the secondary antibody has an
enzyme, such as alkaline phosphatase, attached to it. After the
matrix is washed, a colorless substrate is added.
If the secondary antibody has bound to the primary one, the
colorless substrate is hydrolyzed by the attached enzyme &
produces a colored compound that accumulates at the site of
action.

45. Seek Regulatory and Public Approval.
• Develop Food Safety Data
• Design Reliable Environmental Safety Measures
• Effectiveness & Target Animal Safety Data
• Convince Regulatory Agencies (CVM and Foreign)
• Convince Producers and Customers to Buy

46. PATTERNS AND
INHERITENCE OF TRANS
GENES
 Extra chromosomal
 Degradation
 Integrated at multiple sites
 (Transgenic) X (non-trangenic)
 20-22 % in F1 (as extra chromosomal)
 50% in F2

47. References:
Walker J.M., Gingold E.B, “Molecular Biology &
Biotechnology”, 2nd edi, 1993, panima publishing
educational book agency, New Delhi, 144.
MOLBIO: fundamentals of molecular biology”, 1st
edi. 2005, Himalaya publishing house, Meerut, page
no.-20-28.
http://www.web-books.com/MoBio/Free/Ch9B.htm
http://en.wikipedia.org/wiki/CDNA_library
http://www.molecular-plant-
biotechnology.info/molecular-probes-and-gene-
libraries/construction-and-screening-of-genomic-
and-cDNA-libraries.htm

48. ANY QUESTIONS??