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Genetic engineering in animal cells

Seyed Mohammad Motevalli

October 2013

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OutlineDefinition Historical backgroundGeneral strategyMethods of creation of transgenic animalsDNA microinjectionRetrovirus-mediated gene transferEmbryonic stem cell-mediated gene transfer

How can we get embryonic cells?Selection of transfected cellsGene expressionTransient gene expressionStable gene expression

Transgenic Animals as Biotechnology

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DefinitionThe term transgenic animal refers to an animal in which

there has been a deliberate modification of the genome, the material responsible for inherited characteristics, in contrast to spontaneous mutation (FELASA September 1992, revised February 1995).

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Historical background

During the 1970s, the first chimeric mice were produced (Brinster, 1974)

 DNA microinjection, the first technique to prove successful in mammals, was first applied to mice (Gordon and Ruddle, 1981)

 retrovirus-mediated transgenesis (Jaenisch, 1976)embryonic stem (ES) cell-mediated gene transfer (Gossler

et al., 1986) the term transgenic was first used by J.W. Gordon and

F.H. Ruddle (1981) in 1981

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General strategyIsolation and cloning of the gene of interestGeneration of a (expression-competent) DNA-construct or genetically

modified ES cellsIsolation of oocytes or blastocystsInjection of DNA or genetically modified ES cells and transfer of the

injected embryos into a foster mouseAnalysis of the off-spring Analysis of the expression and biological effects

of the gene of interest in the transgenic animals

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Methods of creation of transgenic animalsTransfer of ‘naked DNA’Calcium phosphate method

DEAE-dextran

Encapsulation of DNA in liposome vesicles

Electroporation

Micro-injection

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Methods of creation of transgenic animalsDNA microinjectionTransfection – transfer of nucleic acid into mammalian

cells (also refers to DNA-mediated transformation)insertion of DNA directly into the nucleus of individual

cells

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Methods of creation of transgenic animalsDNA microinjection

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Methods of creation of transgenic animalsDNA microinjection

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Methods of creation of transgenic animalsDNA microinjection

Less than 5% of the microinjected fertilized eggs become transgenic progeny

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Methods of creation of transgenic animalsViral-assisted DNA transfer Simian virus SV40Retrovirus-mediated gene transfer

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Methods of creation of transgenic animalsRetrovirus-mediated gene transfer

Retroviral vectors can be used to create transgenic animals

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Methods of creation of transgenic animalsRetrovirus-mediated gene transfer

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Methods of creation of transgenic animalsEmbryonic stem cell-mediated gene transferEmbryonic stem cellsFormation of gametocytesInjection into blastocystsInjection into foster motherFormation of new individual

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How can we get embryonic cells?

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Selection of transfected cells

Cell selection by genetic markers

proportion of cells that accept exogenous DNA may be as low as 1 in 106

marker gene incorporated into a recipient cell, allows successful transfectants to be identified

marker can be on the same vector as target gene

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Selection of transfected cells

Non-dominant genes

target cells must have endogenous genes mutated or removed, only works with mutant cells Dominant genes

selection of any cell type, those that acquire specific gene during transfection will surviveAntibiotic resistance genes

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Gene expression Transient gene expression

cells are not genetically stable and soon lose their expression ability

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Gene expression Stable gene expression

DNA is integrated into the nucleus of the host cell, or efficient extrachromosomal replication system is maintained during cell division