Transgenic animals

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Introduction : 

Introduction Ability to introduce/delete functional genes into animals Mouse continues to serve as a starting point for implementing gene transfer procedures Results obtained in mouse are different in other species

Reasons for producing transgenic animals : 

Reasons for producing transgenic animals Obtain fundamental information on living organisms and human diseases Use animals as a source of pharmaceutical proteins Improve animal production

Historical background : 

Historical background In 1977 Gurdon transferred mRNA and DNA into Xenopus eggs In 1980 Brinster et al. used fertilized mouse ova Gordon and Ruddle, 1981 coined the term “transgenic “ animals carrying new genes

Slide 5: 

Definition: introduction of exogenous DNA sequence into the genome of a pluricellular organism and is transmitted to progeny GMO

Methods : 

Methods DNA microinjection ES cells Retroviral mediated gene transfer Gene transfer using sperm Nuclear transfer (cloning)


DNA MICROINJECTION GENERAL METHODS: Preparation of DNA for microinjection Superovulation Harvesting superovulated eggs Microinjecting Ova reimplantation Mating of transgenic mice Visual identification of transgenic mice

Preparation of DNA for microinjection : 

Preparation of DNA for microinjection Low-ionic microinjection buffer (10mM tris; pH 7.4; 0.1-0.3 mM EDTA) DNA concentration between 1.0 and 3.0 ng/ml Dissimilar ends are more efficient than blunt ends Construction and isolation of DNA DNA purification Quantification of DNA

The Genetically Altered Mouse : 

The Genetically Altered Mouse Advantages: --attains sexual maturity rapidly (4-6 wks) --short gestation (19-21 d) --most genetic information/sequencing known --availability of inbred strains --established embryonic stem cells Disadvantages: --size --functional/physiological models in other rodents --variety of infectious diseases --genetic variability and misuse --nomenclature and numbers of available strains Disadvantages

Superovulation : 

Superovulation Strain: Hybrid animals C57BL/6 inbred strain is generally used Isogenicity and homogenicity

Superovulation : 

Superovulation Induction: - PMSG (pregnant mare’s serum gonadotropin) (mimic FSH) and HCG (human chorionic gonadotropin) (mimic LH) - L:D 12-14h - PMSG at noon followed after 48h with HCG 20-30 eggs per female - 5.0-7.5 IU per female (3-8 weeks)

Harvesting of superovulated eggs : 

Harvesting of superovulated eggs Mating (late afternoon following the administration of HCG) Fertilized embryos used for microinjection are 0.5 days post coitum Egg recovery Storage in a incubator chamber

Microinjection : 

Microinjection Egg selection 60-120 eggs per hour

Ova reimplantation : 

Ova reimplantation Female mice are maintained and observed for visual evidence of proestrus Mating to vasectomized males Pseudopregnant females Surgically transfer of 20-25eggs

Strategy to obtain pseudopregnant females : 

Strategy to obtain pseudopregnant females Random mating Mating each day females which are in estrus Superovulation to induce estrus

Mating transgenic mice : 

Mating transgenic mice Founder transgenic mice are mated to non transgenic partners Intercrosses between hemizygous mice within families

Visual identification : 

Visual identification Elastase-ras Tyrosinase minigene

Transgenic phenomenology : 

Transgenic phenomenology Transgene expression Genetic mosaicism Identification of mosaic animals Intrafamily variation in expression Sex chromosome Multiple sites

Slide 31: 

Homozygous vs. hemizygous Genetic instability Transgenic production

Slide 32: 

Homozygous vs. hemizygous

Metodology: : 

Metodology: 1) gene knock-out 2)gene knock-in 3)double replacement approach

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