User talk:Z3254598

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In group with z3288970/3217686/3332227

Lab 5

transgenic : random chromosomal integration of foreign DNA

homologous recombination ->site directed disruption (knockout) or replacement with a modified variant of a gene allele (knock-in) ->conditional KO/KI tissue specific and inducible

TG: take fertilised egg and inject dna into male nucleus, and the dna will randomly insert into the genome, gene gets expressed and you produce more of the protein. altho inserted randomly, doesnt often effect other parts of genome. You then take this and insert into a pseudopregnant female types of transgenic: target protein overexpressed->>more than normal may be ectopically expressed-->protein expressed in tissues which do not normally express it may be mutated protein to produce: constitutively active (gain of function) or dominant negative (loss of function) form of a protein or to mimic a mutated protein observed in a human genetic disease.

because of random insertion, each resultant foudner contains the transgene in a different site in the genome -this is not normally a problem, however the position effect can profoundly affect the expressino of both the transgene and the endogenous genes whos regulatory elements may be disrupted. -transgene may disrupt the coding region of endogenous genes (insertional mutagenesis) confounding the phenotype

therefore it is essential that lines from several different founders lines be examined beofre a conclusion relating a specific phenotype to transgene expression is made.

to assess dose response relatinoships b/w transgene expression and phenotype it is also important to assess lines of mice which express the transgene at different levels.

KO/KI mice def: a mouse in which a specific mouse gene has been gentically modified and the modification is transmitted thru the germ line.

KO: is a mod in which the activity of the gene is eliminated [deleting the gene/key reigon] KI: is a mod in which a specific mutation(s) or rearrangement is introduced and the gene remains functional.

Why make Ko/Ki? to understand gene function/create mouse models to study pathophysiology of disease adn test therapeutic approaches to disease/most useful to mimic recessive disorders (loss of function mutations)

A fragment of genomic DNA is introduced into a mammalian cell and it can locate and recombine with the endogenous homologous sequences. this type of homologous recombination is also commonly referred to as gene targeting -occurs in yeast,bactera and cetain viruses however it is a rare event in mammalian cells except germ cells.

How to make a KO mouse: 1. Pluripotent embryonic stem (ES) cells Get cells, grow them in culture, then you electroporate so that the cell membranes become leaky, allowing for dna to pass thru. You select the dna you wish to target. 1. es cells isolated 2. dna targtting construct into the ES cells 3. microinjection microscope use to take up es cells with syringe 4. genetically modified ES cells injected into blastocysty 5. blasotcysty implayed into surrogate mother -- 2. Construction of KO/KI DNA vector Number of markers/selection procedures to make sure the cells you inject back into the blastocyst has the correct genome

KO: advantage: specific insertion of gene at specific location ro removal of specific gene mimic recessive disorders (loss of function mutations) disadvantage: low level of ES cells with wanted gene inseerted further breeeding necessary to obtain non-chimeric homozygotic animal

TG: advantage: relative high rate of insertion of the injected gene into the genome use for dominant disorder

disadvantage: random inseration can lead to position effects