PCR Applications in Farm Animals

Introduction:- What is The Polymerase Chain Reaction? It’s a means of selectively amplifying a particular segment of DNA. The segment may represent a small part of a large and complex mixture of DNAs It can be thought of as a molecular photocopier.

Different Types of PCR TC 412 T 300 TC 312

Invented by Kary Mullis Mullis and Faloona, 1983. Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. First published account appeared in 1985. Nobel Prize in Chemistry 1993

Kary Mullis

PCR (Polymerase Chain Reaction) In vitro amplification of specific DNA fragment. Uses two oligonucleotide primers. Complementary to opposite strands of a duplex DNA. Taq DNA polymerase –Thermus.aquaticus. Thermostable polymerases.

Thermus.aquaticus from hot springs in Yellowstone National Park, USA

How does PCR work?

Advantages & Disadvantages of PCR Approach Easy to pick up contaminants. Cannot discriminate between viable and nonviable or infectious and noninfectious cells or viruses. Subject to inhibition by chemicals in environmental samples. humic substances metals More sensitive and detects all organisms regardless of physiological state. Fast and easy to perform.

Applications of PCR Detection of mutations in DNA. Marker synthesis. Diagnosis – prenatal. DNA profiling in Forensic science (DNA fingerprinting). Sex Determination in animals. Determination of evolutionary relationships among organisms.

PCR Applications in Farm Animals Sex Determination of Prenatal Cells. Bovine Carcass Sexing.

1- Sex Determination of Prenatal Cells Determining sex of fetuses at risk for X-linked inherited disorders. (a) Oocytes are removed from the mother following superovulation and fertilized in vitro. (b) The oocytes that are fertilized successfully are cultured in vitro until there are 6 to l0 cells in each embryo. (c) A hole is made in the zona pellucida and a single cell removed from each embryo. (d) Amplification of the DYZ.l sequence is attempted. (e) Only in DNA from males is the male-specific DYZl sequence amplified by PCR, giving rise to a 149-bp, male-specific fragment. The lane marked with the male symbol is a positive control showing the expected fragment; the lane marked B (for "Blank") is from a PCR that included all the reagents but no DNA and is used to detect any contamination. Female embryos are negative (lanes 1, 2, and 5) and are implanted into the mothers.

2- Bovine Carcass Sexing

Transgenic Animals Gene transfer is transplantation of specific genes from one individual to another using laboratory techniques. The generation of transgenic animals is one of the most complex aspects of genetic engineering, in terms both of :- (1) Technical difficulty. (2) The ethical problems that arise.

History of transgenic animals 1966 First report of microinjection of mouse embryo. 1974 First transgenic integration via viral infection. 1981 First transgenic mouse (Gordon, J & Ruddle, F) 1982 Production of ”Super mouse” carrying GH. 1983 Tissue specific gene expression in transgenic mice. 1984 Transgenic dwarf mice carrying hGH grow to normal size. 1987 Transgenic pig, sheep, rabbit and fish. 1988 First patented transgenic mouse - ”Oncomouse” 1991 First report on microinjection for transgenic goat. 1996 ES cells used for nuclear transfer sheep. 1997 Somatic cell from adult sheep used for cloning by nuclear transfer.

Mammal Cloning Timeline 1984 – A live lamb was cloned from sheep embryo cells. 1986 – Early embryo cells were used to clone a cow. 1993 – Calves were produced by transfer of nuclei from cultured embryonic cells. 1995 – Two sheep, named Megan & Morag, were cloned using embryo cells. 1996 – Birth of Dolly, the first organism to be cloned from a fully differentiated adult cell. 1997 – Transgenic sheep named Polly was cloned containing a human gene. Megan and Morag Dolly

1998 – 50 mice were cloned in three generations from a single mouse. 1998 – 8 calves were cloned from a single adult cow, but only 4 survived to their first birthday. 1999 – A female rhesus monkey named Tetra was cloned by splitting early embryo cells. 2000 – Pigs and goats reported cloned from adult cells. 2002 – Rabbits and a kitten reported cloned from adult cells.

Methods DNA Microinjection of fertilized eggs. Embryonic stem cell injection in blastocysts. Retroviral. Nuclear transfer.

DNA Microinjection Method outline : Stimulate donor females to superovulate  Increase in egg production (from 5-10 to 35). The superovulated females are mated and the fertilized eggs are obtained. Immediate microinjection of DNA into the male pronucleus. Microsurgical implantation of 25-40 eggs into a pseudopregnant foster mother ( mated to a vasectomized male). After three weeks pups are born (~25% transgenic).

Tracy produced a very valuable drug in her milk This sheep is called Tracy She was born in 1990 She produced large quantities of a very valuable protein called AAT (alpha antitrypsin) in her milk. AAT can be used to treat the symptoms of Cystic Fibrosis and Emphysema.

Methods DNA Microinjection of fertilized eggs. Embryonic stem cell injection in blastocysts. Retroviral. Nuclear transfer.

Embryonic stem cell injection in blastocysts Objectives: - specific gene modification or inactivation. - gene repair. Steps: - Construct vector. - Introduce into ES cells. - Screen for target event. - Inject ES cell clone into blastocysts. - Transfer to pseudo-pregnant recipient.

Microinjection in fertilized eggs Holding pipette Injection pipette

Microinjection of ES cells in blastocysts 2

Microinjection of ES cells in blastocysts 3

Methods DNA Microinjection of fertilized eggs. Embryonic stem cell injection in blastocysts. Retroviral. Nuclear transfer.

Retroviral Vector Method Retroviral vectors infect the cells of an early-stage embryo prior to implantation into a receptive female. Advantage: Effective to integrate the transgene into the genome. Drawbacks: The vector can only transfer small DNA fragments (~8kb). Virus DNA can also be integrated. This method is rarely used for creating transgenic animals that have a commercial end use.

Methods DNA Microinjection of fertilized eggs. Embryonic stem cell injection in blastocysts. Retroviral. Nuclear transfer.

Method of Nuclear Transfer in Livestock

Somatic Cell Nuclear Transfer (SCNT) Starts with removal of polar body and chromosomes from an oocyte. Donor cell then inserted into perivitelline space of enucleated oocyte.

(SCNT) Oocyte and donor cell are fused and activated by an electric pulse to begin cell division. Developed embryos transferred to surrogate females. Birth of an individual.

Benefits Science Agriculture Medicine Industry Basic research. Increased yields, faster than conventional breeding. Increased quality (nutritional content, product characteristics, etc.) Disease resistance. Medicine Pharmaceuticals (Pharming). Gene therapy. Industry Enzymes. Raw materials. New substances.

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