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Hands on training in Animal Biotechnology

at

Guru Angad Dev Veterinary and Animal Sciences University


(GADVASU)
Ludhiana (Punjab)
Duration
1 June to 14 July, 2016

Copyright 2013-2014
Presented By:
Shivani Guleria
(301501016)
Introduction
Guru Angad Dev Veterinary and Animal Sciences University
(GADVASU)
Established in 6 april,2006 after carving out from the Punjab
Agricultural University(PAU).
Situtated in Ludhiana (Punjab)
Priority research areas of the School of Animal Biotechnology are:

Molecular
Animal diagnostics Stem and
genomics & cell biology

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Vaccinology
Animal biotechnology is a branch of biotechnology in which molecular
biology techniques are used to genetically engineer (i.e. modify the
genome of) animals.
To improve their suitability for pharmaceutical, agricultural or
industrial applications.[1]
Examples:
Production human pharmaceutical proteins include enzymes, clotting
factors, albumin and antibodies.
Increased milk and meat production in animals

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Contents
1. Genomic DNA isolation from Blood

2. RNA isolation from a tumor tissue of canine

3. Chicken Embryo Fibroblast Primary cell culture

4. Virus Transfection in cell Culture

5. GFP Expression in HeLa Cells

6. Immunohistochemistry

7. Western Blotting

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8. Protein Expression in bacterial (E. coli) system and their detection
by SDS-PAGE
9. References
Genomic DNA Isolation
Method- Phenol- chloroform
Source Blood of buffalo (Bubalus bubalis )
Result:

Shear DNA

Genomic DNA

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Fig 1: (a) Isolated Genomic DNA ( b) Qualitative Analysis (C) Quantitative Analysis
From Blood Bubalus bubalis (Agarose Gel Electrophoresis ) (Nanodrop method) 260/280= 1.8
RNA isolation
Method Trizol method
Source- Tumor tissue of canine
Result:

Fig 2: Qualitative analysis


(Agarose Gel Electrophoresis)

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Quantitative analysis:
260/280 ratio = 1.36
Conc.= 6.32 ng /ml.
Chicken Embryo Fibroblast cell culture
Basic idea is to break any tissue into single cells by proteolytic action of
trypsin and then suspend these cells into growth medium for cell sheet
formation.

Embryo

Result:
Fibroblastic cells are bipolar or multi-polar, have elongated shapes and grow
attached to a substrate.

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Fig 3 :Chicken embryo fibroblast cell monolayer
Virus Transfection in cell Culture

The cytopathic effect of Bovine Herpes Virus on Vero cells


The changes in cell morphology caused by infecting virus are called
cytopathic effects (CPE).
1. Rounding of the infected cell.
2. Syncytia Formation.
3. Enlargement of cell.

Fusion of cell

Enlargement of cell
Cell enlargement

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Fig 4 : Cytopathic effect of virus
GFP Expression in HeLa Cells

Stable Expression: The foreign gene becomes part of the genome and is
therefore replicated. Descendants of these transfected cells, will also
express the new gene, resulting in a stably transfected cell line.
GFP is a Green Fluorescent Protein that exhibits bright green fluorescence
when exposed to light in the blue to ultraviolet range.
Isolated from Jelly fish Aequorea victoria
Result:

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Expressed
GFP protein

Fig 5 :GFP expression in HeLa cells


ImmunoHistochemistry
It is used for the localization of an antigen in a tissue using a specific
antibody.
Detection of PCNA marker in Goat mammary tissue.
Antibody :Anti-PCNA antibody (PC 10)
Result:

Cells that doesnt contain PCNA marker


(purple stain)

Cells containing PCNA marker


(brown stain)

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Fig 6: Detection of PCNA marker
Western Blotting

This technique is used to detect specific proteins in a mixture of protein


sample.
Technique uses three step:
Separation by sizeTransfer to a solid supportMarking target protein
using a proper primary and secondary antibody to visualize.
Protein used: Recombinant Dog Protein expressed in E.coli.
Result:

Bands

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Nitrocellulose membrane

Fig 7:Transfer and detection of Protein bands on nitrocellulose membrane


Protein Expression in bacterial (E. coli) system
and their detection by SDS-PAGE

BL21 E.coli Competent cells were used


(Gene- PCV ORF 2 gene is inserted in expression vector (pET 302NT His)

1hr 2hr 3hr 4hr 5hr

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Fig8: Protein expression study at different interval of time
Refrences
1. http://www.nature.com/subjects/animal-biotechnology
2. http://www.gadvasu.in/college-matter.asp?page=about%2Dcollege
3. Laemmli U K (1970) .Cleavage of structural protein during the assembly of the head
bacteriophage T4. Nature ,227, 680-688
4. Sambrook J and Russell D W(2001) Molecular Cloning: A Laboratory Manual. (3rd ed) Cold
Spring Harbor Laboratory, Cold Spring Harbor, New York.
5. Wilson K, Walker J. Principles and techniques of biochemistry and molecular biology.
Seventh Edition.pp 399-407
6. Renart J, Reiser J, Stark GR (1979)Transfer of proteins from gels to diazobenzyloxymethyl-
paper and detection with antisera: a method for studying antibody specificity and antigen
structure,Proceedings of the National Academy of Sciences USA 76 (7): 31163120
7. Towbin H, Staehelin T, Gordon J (1979)Electrophoretic transfer of proteins from
polyacrylamide gels to nitrocellulose sheets: procedure and some applications, Proceedings
of the National Academy of Sciences USA 76 (9): 43504354
8. Yalow, Rosalyn S.; Berson, Solomon A. (1960)Immunoassay of endogenous plasma insulin in
man, The Journal of Clinical Investigation 39: 11571175

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Thank you

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