CHARACTERIZATION OF TRANSGENIC

FISH

By: Saadia Laraib

Roll no. 306th Semester (A)

Steps for Making Transgenic Fish

Step 1. Decide Gene/Protein to Add.

Step 2. Decode Protein and Translate to cDNA Code.

cDNAProtein

Steps for Making Transgenic Fish

Step 3. Prepare Gene Construct.

Protein Gene Promoter Gene Gene Construct

Steps for Making Transgenic Fish

Step 4. Insert Construct into Bacterial Plasmid.

Steps for Making Transgenic Fish

Step 5. Insert Plasmid inBacterial Strain & MakeBillions of Copies.

Steps for Making Transgenic Fish

Step 6. Isolate Plasmids from Bacteria and Cleave into Linear Cassettes.

Steps for Making Transgenic Fish

Step 7. Insert Over 1 Million Cassettes into Each Newly Fertilized Egg.

Steps for Making Transgenic Fish

Step 8. Incubate and Grow Out Surviving Fry.

Steps for Making Transgenic Fish

Step 9. Find the Transgenics and Select Fish(s) with Desired Characteristics.

Steps for Making Transgenic Fish

Step 10. Breeding Program to Stabilize Transgene.

SOUTHERN BLOTTING1. Extract and purify DNA from cells2. DNA is restricted with enzymes3. Sort by electrophoresis4. Denature DNA5. Transfer to nitrocellulose paper6. Block with excess DNA7. Wash off unbound probe8. Autoradiograph

? copies of gene X

extract

DNA

Looking for Gene X

Step 1. Restriction Enzyme Digestion

EcoR I EcoR I EcoR I EcoR I

Step 1. Restriction Enzyme Digestion

Step 2. Gel Electrophoresis

_ +

Step 2. Gel Electrophoresis

_ +

Step 2. Gel Electrophoresis

Goals of Southern Hybridization

Immobilize DNA onto a permanent substrate

‘Membrane’paper-like matrixnylon or nitrocelluloseusually has a slight positive charge

Step 3. DNA Denaturation

T G A A TC

A C AT T G

• Eliminate hydrogen bonds with sodium hydroxide (NaOH)

Step 4. Transfer DNA to Membrane

Step 6. Pre-hybridization

Prehybridization bufferscontain ‘blocking reagents’that occupy available binding sites on the membrane

Step 7. Hybridization

Step 7. Hybridization

Step 8. Washes

Step 9. Anti-DIG

Step 9. Anti-DIG

Step 10. Washes

Step 11. CSPD

Step 12. Detection DIG-labeled probes

emitting minute amounts of light (chemiluminescence)

32P-labeled probes emitting ß-particles

Step 12. Detection DIG-labeled probes

emitting minute amounts of light (chemiluminescence)

32P-labeled probes emitting ß-particles

Autoradiography film can detect this radiation

Results: How many copies of

‘Gene X’ does the fish possess?

3

Dot Blot/ Northern Blotting

 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 

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.

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. 

RT-PCRDetection of Specific RNAs

Annealing of Downstream Primer to RNA

Reverse Transcription With AMV Reverse Transcriptase

RNA Copied From 3’ to 5’ into cDNA

Amplification of cDNA by PCR

SCREENING BY IMMUNOLOGICAL ASSAY:

 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.

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

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

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.htmhttp://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 

ANY QUESTIONS??