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2-8-11 Site-Spec. Rec. _ Transposition I

2-8-11 Site-Spec. Rec. _ Transposition I - Conventional...

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1 Conventional gene targeting - for knockout of the mouse HPRT gene on chromosome X Mouse embryonic stem (ES) cells are transfected with a linear targeting vector. It contains Neo - a “positive selection marker” (antibiotic resistance gene), flanked by arms of DNA with homology (sequence identity) to part of the chromosomal target locus – the HPRT gene. Homologous recombination between the vector and chromosomal target locus can result in replacement of chromosomal HPRT sequence with the Neo marker. The HPRT gene is disrupted, and the cell is now resistant to neomycin (G418). Making mice with a targeted gene change Targeted embryonic stem cells are injected into blastocyst-stage embryos , which can then be re-implanted into a female mouse and carried to term.
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2 Making mice with a targeted gene change Female mouse implanted with (ESC-injected) blastocysts gives birth to chimeric F1 mice, some of which have the targeted gene change in their germ line cells. (We fervently hope!) F1 mice that have the targeted mutation in the germ line can be back- crossed to get F2 mice with the mutation in all cells of the animal. The F2 mice will be heterozygous for the mutation. They can be crossed to get mice that are homozygous. Gene targeting has historically been very inefficient In most gene targeting experiments in mouse ES cells, the frequency of targeted events is low : one to a few targeted clones out of every 10 5 to 10 7 cells transfected. The vast majority of transfected clones have instead undergone non-homologous vector integration (NHVI) , i.e., integration of the targeting vector at random genomic locations. This makes it hard to isolate of targeted clones of ES cells; it’s often necessary to laboriously screen hundreds or thousands of transfected ES cell clones. Clever selection schemes can reduce this problem, but not eliminate it altogether (see next figure). Early attempts with human stem cells fared no better. Gene targeting - positive/negative selection In this scheme, the targeting vector also includes a negative selection marker , HSV - TK (the Herpes simplex virus thymidine kinase gene). The TK gene will typically not be integrated into the target locus by HR. Random integration of the vector (NHVI) often includes the TK gene. These cells can be selectively killed using a toxic analog of thymidine, FIAU . It's not perfect, but it does help reduce the background of NHVI, so that fewer clones must be screened to find the targeted ones. Chromosomal Target Gene Vector ends invade the homologous chromosomal target locus, and set up heteroduplex intermediates that must be resolved as crossovers to bring about an exchange of vector sequence for chromosomal. This Vector-Invades-Chromosome mechanism is extremely inefficient, because crossover is not the preferred pathway of resolution in mitotic cells!
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2-8-11 Site-Spec. Rec. _ Transposition I - Conventional...

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