logging in or signing up Transgenic animals dagaro2009 Download Post to : URL : Related Presentations : Share Add to Flag Embed Email Send to Blogs and Networks Add to Channel Uploaded from authorPOINT lite Insert YouTube videos in PowerPont slides with aS Desktop Copy embed code: (To copy code, click on the text box) Embed: URL: Thumbnail: WordPress Embed Customize Embed The presentation is successfully added In Your Favorites. Views: 4109 Category: Education License: All Rights Reserved Like it (10) Dislike it (1) Added: February 11, 2009 This Presentation is Public Favorites: 5 Presentation Description No description available. Comments Posting comment... By: quemd (7 month(s) ago) thank u Saving..... Post Reply Close Saving..... Edit Comment Close By: PraveenKumarPK (13 month(s) ago) thank u Saving..... Post Reply Close Saving..... Edit Comment Close By: PraveenKumarPK (13 month(s) ago) thank u Saving..... Post Reply Close Saving..... 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See all Premium member Presentation Transcript TRANSGENIC ANIMAL TECHNOLOGY : TRANSGENIC ANIMAL TECHNOLOGY 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 : 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 You do not have the permission to view this presentation. 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Transgenic animals dagaro2009 Download Post to : URL : Related Presentations : Share Add to Flag Embed Email Send to Blogs and Networks Add to Channel Uploaded from authorPOINT lite Insert YouTube videos in PowerPont slides with aS Desktop Copy embed code: (To copy code, click on the text box) Embed: URL: Thumbnail: WordPress Embed Customize Embed The presentation is successfully added In Your Favorites. Views: 4109 Category: Education License: All Rights Reserved Like it (10) Dislike it (1) Added: February 11, 2009 This Presentation is Public Favorites: 5 Presentation Description No description available. Comments Posting comment... By: quemd (7 month(s) ago) thank u Saving..... Post Reply Close Saving..... Edit Comment Close By: PraveenKumarPK (13 month(s) ago) thank u Saving..... Post Reply Close Saving..... Edit Comment Close By: PraveenKumarPK (13 month(s) ago) thank u Saving..... Post Reply Close Saving..... 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See all Premium member Presentation Transcript TRANSGENIC ANIMAL TECHNOLOGY : TRANSGENIC ANIMAL TECHNOLOGY 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 : 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