Transgenic creatures and knockout creatures.


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3 primary approaches to do natural research:Do research in test tubes.Do research with cells.Do examine specifically with creatures.. Transgenic creatures and knockout animalsPart 1: Transgenic animals:Introduction to transgenic animals.How to make transgenic animals?How to make restrictive transgenic animals?Applications of transgenic animals.Part 2: Knockout animalsIntroduction to knockout animals.How to mak
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Transgenic creatures and knockout creatures

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3 fundamental approaches to do organic examination: Do scrutinize in test tubes. Do inquire about with cells. Do examine specifically with creatures.

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Transgenic creatures and knockout creatures Part 1: Transgenic creatures: Introduction to transgenic creatures. How to make transgenic creatures? How to make restrictive transgenic creatures? Utilizations of transgenic creatures. Section 2: Knockout creatures Introduction to knockout creatures. How to make knockout creatures? How to make restrictive knockout creatures? Utilizations of knockout creatures.

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Transgenic Animal has one or more remote qualities in serted into chromosome DNA inside its cells falsely . In the wake of infusing remote quality into the pronucleus of a treated egg or blastocyst, outside quality is in serted in an arbitrary manner into chromosome DNA: Random ly ( Foreign quality may disturb an endogenous quality critical for ordinary improvement , and the chance is around 10%. ) different duplicates

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Transgenic creatures and knockout creatures Part 1: transgenic creatures: Introduction to transgenic creatures. How to make transgenic creatures? How to make restrictive transgenic creature? Uses of transgenic creatures. Section 2: Knockout creatures Introduction to knockout creatures. How to make knockout creatures? How to make contingent knockout creatures? Uses of knockout creature.

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ES cell change Injection of quality into prepared egg

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Method 1: ES cell change versus Strategy 2: Injection of quality into prepared egg 1. ES cell change functions admirably in mice as it were. Other transgenic creatures are delivered by egg infusion 2. ES cell change gives more control of the combination step (determination of steadily transfected ES cells) 3. Infusion of quality into treated egg is less solid (reasonability of eggs, recurrence of combination), however it serves to dodges chimeric creatures

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Injecting prepared eggs The eggs are reaped from mice (superovulated or characteristic matings) . The DNA is normally infused into the male pronucleus . The eggs can be moved around the same time (1 cell) or the following day (2-cell s ) into pseudopregnant female oviducts .

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Breeding T ransgenic creatures (transgenic organizers ) Transgenic creatures Individually are backcrossed to non-transgenic creatures. Don\'t intercross diverse organizers . E ach organizer comes about because of a different RANDOM transgene incorporation even t.

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Transgenic creatures and knockout creatures Part 1: transgenic creatures: Introduction to transgenic creatures. How to make transgenic creatures? How to make contingent transgenic creatures? Uses of transgenic creatures. Section 2: Knockout creatures Introduction to knockout creatures. How to make knockout creatures? How to make restrictive knockout creatures? Uses of knockout creature.

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Conditional Transgenic mouse The statement of transgene in transgenic mouse can be incited

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Important Considerations for Conditional Transgenes have low or no expression when not actuated Large contrast amongst initiated and non-prompted quality expression Transgene expression quickly turns on or off. Inducer (doxycycline, tamoxifen, cre) is not harmful and effectively directed

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Tetracycline Controlled Transactivator tTA "Tet-off" tet R VP16 Doxycycline squares tTA DNA restricting tTA ties to tetO to enact interpretation

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Reverse Tetracycline Controlled Transactivator tTA "Tet-on" rtet R VP16 Doxycycline permits rtTA to tie to tetO Without doxcycline rtTA can not tie to tetO

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Tetracycline Regulation: Summary No Doxycycline Doxycycline tTA communicated not communicated rtTA not communicated

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Transgenic creatures and knockout creatures Part 1: transgenic creatures: Introduction to transgenic creatures. How to make transgenic creatures? How to make restrictive transgenic creature? Uses of transgenic creatures. Section 2: Knockout creatures Introduction to knockout creatures. How to make knockout creatures? How to make contingent knockout creatures? Uses of knockout creature.

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Applications of Transgenic Animals Transgenic mice are frequently produced to 1. portray the capacity of a promoter to direct tissue-particular quality expression e.g. a promoter can be joined to a correspondent quality, for example, LacZ or GFP 2. analyze the impacts of overexpressing and misexpressing endogenous or remote qualities at particular times and areas in the creatures 3 Study quality capacity Many human ailments can be demonstrated by bringing the same change into the mouse. In place creature gives a more finish and physiologically important photo of a transgene\'s capacity than in vitro testing . 4. Drug testing

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Example 1: Transgenic Cattle Cloned transgenic cows produce milk with more elevated amounts of beta-caein and k-casein Published in Nature, Jan, 2003

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Example 2: Transgenic Mouse The development hormone quality has been designed to be communicated at abnormal states in creatures. The outcome: BIG ANIMALS Mice bolstered with overwhelming metals are 2-3 times bigger M etallothionein promoter directed by substantial metals

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Example 3: Transgenic Mouse Trangenic mouse incipient organism in which the promoter for a quality communicated in neuronal forebears (neurogenin 1) drives articulation of a beta-galactosidase journalist quality. Neural structures communicating the journalist transgene are dull blue-green.

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Example 4: GFP transgenic mouse (Nagy) 9.5 day fetuses - GFP and wt Tail tip

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GFP transgenic mouse (Nagy)

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Example 5: Wild and household trout react diversely to overproduction of development hormone. In this way, GH is not viable to local trout .

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Example 6: Transgenic mice as instruments Normal mice can\'t be contaminated with polio infection. They do not have the cell-surface Polio infection receptor . Be that as it may, human has Polio infection receptor . T ransgenic mice communicating the human quality for the Polio receptor can be tainted by polio infection and even create loss of motion and other obsessive changes normal for the malady in people

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Transgenic creatures and knockout creatures Part 1: transgenic creatures: Introduction to transgenic creatures. How to make transgenic creatures? How to make restrictive transgenic creature? Uses of transgenic creatures. Section 2: Knockout creatures Introduction to knockout creatures. How to make knockout creatures? How to make contingent knockout creatures? Uses of knockout creatures.

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thump out Animal One endogenous quality in a creature is changed. The quality can not be communicated and loses its capacities. DNA is brought first into embryonic stem (ES) cells. ES cells that have experienced homologous recombination are recognized . ES cells are infused into a 4 day old mouse developing life : a blastocyst . Knockout creature is gotten from the blastocyst.

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Transgenic creatures and knockout creatures Part 1: transgenic creatures: Introduction to transgenic creatures. How to make transgenic creatures? How to make contingent transgenic creature? Utilizations of transgenic creatures. Section 2: Knockout creatures Introduction to knockout creatures. How to make knockout creatures? How to make restrictive knockout creatures? Utilizations of knockout creatures.

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Vector plan Recombinant DNA techniques: Simple KO Structural quality fancied (e.g. insulin quality) to be "knocked out" is supplanted mostly or totally by a positive choice marker to thump out the quality capacity s. Vector DNA to empower the atoms to be embedded into host DNA particles

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KNOCKOUT MICE Isolate quality X and addition it into vector. Inactivate the quality by embeddings a marker quality that make cell impervious to anti-microbial (e.g. Neomycin) Normal (+) quality X Genome Defective (- ) Gene X Transfer vector with (- ) quality X into ES cells (embryonic undifferentiated organisms) VECTOR e.g.(NeoR) MARKER GENE

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Vector and genome will recombine by means of homologous arrangements Genomic quality Exon 4 Exon 2 Exon 3 Exon 1 Homologous recombination and quality disrution Grow ES cells in anti-microbial containing media; Only cell with marker quality ( without ordinary target quality) will survive

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Solution: Replacement vectors The thump out develop contains the 1) NeoR quality flanked by 2) two portions of the objective quality and 3) the HSVtk quality Part of the quality supplanted with NeoR ES cells are chosen for combination of NeoR and against incorporation of HSVtk* (NeoR+/HSVtk-) on gancyclovir Problems with homologous recombination Unwanted irregular non-homologous recombination is extremely visit. This strategy gives no determination against it

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Homologous recombination Random reconciliation Neo R Neo R+/HSVtk - Neo R+/HSVtk + HSVtk will change over gancyclovir into a poisonous medication and murder HSVtk+ cells Replacement vectors Gene portion 1 Gene section 2 Neo R Linearized substitution plasmid HSVtk

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Typical KO vector *tk:thymidine kinase

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Inject ES cells with (- ) quality X into early mouse developing life Transfer fetuses to surrogate moms Resulting chimaras have a few cells with (+) quality X and (- ) quality X. Mate them with ordinary mice It is fortunate, if germline contain (- ) quality X Screen pups to discover -/+ and mate them Next era will part as 3:1 (Mendelian)

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Embryonic immature microorganisms Harvested from the inward cell mass of mouse blastocysts Grown in society and hold their maximum capacity to create every one of the cells of the developed creature, including its gametes .

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ES cells developing in society

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ES cells are changed Cultured ES cells are presented to the vector Electroporation punched gaps in the dividers of the ES cells Vector in arrangement streams into the ES cells The cells that don\'t bite the dust are chosen for change usi

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