Steps for Making TransgenicFish Step 1. Decide Gene/Protein to Add. Step 2. Decode Protein and Translate to cDNA Code. cDNA Protein
Steps for Making TransgenicFish Step 3. Prepare Gene Construct. Protein Gene Promoter Gene Gene Construct
Steps for Making TransgenicFish Step 4. Insert Construct into Bacterial Plasmid.
Steps for Making TransgenicFish Step 5. Insert Plasmid in Bacterial Strain & Make Billions of Copies.
Steps for Making TransgenicFish Step 6. Isolate Plasmids from Bacteria and Cleave into Linear Cassettes.
Steps for Making Transgenic FishStep 7. Insert Over 1 Million Cassettes intoEach Newly Fertilized Egg.
Steps for Making TransgenicFish Step 8. Incubate and Grow Out Surviving Fry.
Steps for Making TransgenicFishStep 9. Find the Transgenics and SelectFish(s) with Desired Characteristics.
Steps for Making TransgenicFish Step 10. Breeding Program to Stabilize Transgene.
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
Looking for Gene X extract DNA ? copies of gene X
Step 1. Restriction Enzyme Digestion EcoR I EcoR I EcoR I EcoR I
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 HYBRIDIZATIONThis method also utilizes a DNA probe. This is usedfor colony hybridization.The procedure involved is as follows1. Transfer some of DNA in plaque / colony to anylon / nitrocellulose membrane. Because plaquesare areas of lysed bacteria, the phase DNA isdirectly available & will bind to the membrane whentop of the petridish.This can be achieved by soaking in sodium dodecylsulphate & protease.
2. The DNA on the membrane is denatured with alkali to producesingle strands; deprotenised & is bonded to the membrane bybaking or UV radiation.3. The membrane is then immersed in a solution containing anucleic acid probe, which is usually radioactive & incubated toallow probe to hybridize to its complimentary sequence.4. After hybridization, the membrane is washed extensively toremove unhybridised probe.5. The region where the probe has hybridized is visualized byexposure to X-ray film. 6. A colony on the master plate that corresponds to the regionof a positive response on the X- ray film is identified. Cell fromthe positive colony on the master plate are sub cloned becausethe carry the desired DNA.Thus the identification of gene of interest from the wholegenomic library is achieved.
This method is used when a DNA probe is not available.In this method all the clones of the library are grown separately on aplates. A sample of each colony is transferred to a known positionon a matrix, where the cells are lysed & the released proteins areattached to the matrix.The matrix with the bound proteins is treated with an antibody(primary antibody) that specifically bounds to the protein encodedby the target gene.Following the interaction of primary antibody with the target protein(antigen), any unbound antibody is washed away, and matrix istreated with a second antibody (secondary antibody) that is specificfor primary antibody.In many assay systems, the secondary antibody has anenzyme, such as alkaline phosphatase, attached to it. After thematrix is washed, a colorless substrate is added.If the secondary antibody has bound to the primary one, thecolorless substrate is hydrolyzed by the attached enzyme &produces a colored compound that accumulates at the site ofaction.
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 ANDINHERITENCE OF TRANSGENES 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 publishingeducational book agency, New Delhi, 144.MOLBIO: fundamentals of molecular biology”, 1stedi. 2005, Himalaya publishing house, Meerut, pageno.-20-28.http://www.web-books.com/MoBio/Free/Ch9B.htmhttp://en.wikipedia.org/wiki/CDNA_libraryhttp://www.molecular-plant-biotechnology.info/molecular-probes-and-gene-libraries/construction-and-screening-of-genomic-and-cDNA-libraries.htm