Recombinant DNA Technology CHMI 4226 E Week of April 30, 2009 Functional genomics Transgenic mice...
-
Upload
herbert-todd-washington -
Category
Documents
-
view
214 -
download
0
Transcript of Recombinant DNA Technology CHMI 4226 E Week of April 30, 2009 Functional genomics Transgenic mice...
Recombinant DNA TechnologyCHMI 4226 E
Week of April 30, 2009
Functional genomicsTransgenic miceKnock-out mice
Transgenic mice
• Goal: increase the expression of a particular gene in the mouse, with the aim of determining the effect of the gene product on the animal’s phenotype.
Transgenic mice
• Main steps:– Preparation of the transgene (promoter,
epitope tags, etc).– Injection in mouse fertilized egg– Transplantation into foster mother– Analyze the pups for presence and
expression of the transgene– Analysis of the phenotype
Transgenic mice
Transgenic mice
Transgenic mice
Tail was clipped to obtain material for DNA isolation and testing for the presence of the transgene (PCR).
Transgenic mice Transmission of the transgene
Transgenic mice
IRBP: Inter-photoreceptor retinoid binding protein: expression restricted to the retinal photoreceptor cells.
Exons/intron of -globine: mRNA stabilization anf improvement of exportation of the mRNA from the nucleus to the cytoplasm.
Transgenic mice Careful breeding can lead to
interesting findings
Transgenic mice Random insertion of the transgene in the
genome can complicate things a bit…
Transgenic mice
• Avantages:– Alloes on the study the effect of a gene on an animal.– Gene therapy.
• Disadvantages:– Overexpression of the gene– Random insertion into the genome can cause several
artefacts:• Inactivation of other genes that can be the result of the
phenotype• Variable expression levels of the transgene (e.g. insertion
into heterochromatin: « position-effect variegation »)
Knock-out animals
• Animals in which a specific gene has been inactivated by homologous recombination
• Allows on to determine the effect of the absence of a single gene on the animal’s phenotype.
Knock-out mice
• Main steps:– Preparation of the transgene– Transfection into embryonic stem cells (ESC)
and selection.– Transfer ESC into blastocysts and into foster
mothers– Screening of chimeric mice (mice having a
mixtures of cells in which the gene is inactivated or not)
– Breedings to obtain homozygotes for the mutation (-/-)
Knock-out miceEmbryonic stem cells
Knock-out miceEmbryonic stem cells
Knock-out mice
Viral thymidine kinase: converts ganciclovir into a nucleotide analog, which will then inhibit DNA replication.
Knock-out mice
Knock-out mice Example: Knock-out of the pro-death
gene APAF-1
Cell, Vol. 94, 739–750, September 18, 1998,
Knock-out mice
ESC contain the inactivated gene
Knock-out mice
Knock-out mice In-utéro lethality of APAF-1 -/- mice
Cell, Vol. 94, 739–750, September 18, 1998,
Knock-out mice Effect of the deletion of an important gene
APAF-1: an activator of cell death
Cell, Vol. 94, 739–750, September 18, 1998,
Knock-out mice Effect of the deletion of an important gene
APAF-1: an activator of cell death
Cell, Vol. 94, 739–750, September 18, 1998
Knock-out mice
• Avantages:– Precise insertion of the transgene into the genome via
homologous recombination;– Determination of the effect of the elimination of a
single gene on the animal’s phenotype;
• Disadvantages:– Often without effect (compensatory effect of other
genes)– In-utero lethality of the -/- homozygous pups if the
gene is indispensable to the development of the animal;
– Gene is inactivated in all tissues of the animal;
Cre-Lox (Flox) system
• Allows one to regulate in vivo the expression of a transgene or the deletion of a gene.
Cre-Lox (Flox) system
• Principle:– A lox recombination
sequence is inserted on each side of the DNA to be excised;
– Recombination between two lox sites is triggered by the cre protein. lox P site
Lox P
Lox P
Cre-Lox (Flox) system System of Lasker et al.
Cre-Lox (Flox) system
• Construction of two lines of transgenic mice:– One series of mice which
possess the gene of interest, the promoter sequence of which being separated by a transcription STOP sequence, itself flanked by lox P sites;
– A second line of transgenic mice expressing the cre gene, the expression of which is regulated by a promoter of choice.
Cre-Lox (Flox) system
• The two mice strains are bred together
• The expression of cre in the pups will lead to the excision of the STOP sequence and the expression of the gene of interest.
Cre-Lox (Flox) system Targeted expression (in time and space) of the
gene of interest
Cell, Vol. 87, 1317–1326, December 27, 1996
Cre-Lox (Flox) system Targeted expression (in time and space) of the
gene of interest
Cell, Vol. 87, 1317–1326, December 27, 1996
Cre-Lox (Flox) system Targeted expression (in time and space) of the
gene of interest
Cell, Vol. 87, 1317–1326, December 27, 1996
Cre-Lox (Flox) system Targeted expression (in time and space) of the
gene of interest
Cell, Vol. 87, 1317–1326, December 27, 1996
Cre-Lox (Flox) system Targeted expression (in time and space) of the
gene of interest
Cell, Vol. 87, 1317–1326, December 27, 1996
Cre-Lox (Flox) system Application to the generation of knock-out
mice.
Inserted by homologous recombination in ESC
Cre-Lox (Flox) system Deletion of the neo gene
Cre-Lox (Flox) system Application to the generation of knock-out
mice.
• Application: knock-out of the testosterone receptor gene (AR:androgen receptor)
• Why?– Better understand the role of testosteron in
target tissues– AR: only one copy in males (X chromosome)– By classique knock-out techniques: males
become resistant to testosterone (phenotype: feminized and sterile).
Cre-Lox (Flox) system Application to the generation of knock-out
mice.
13498–13503 PNAS October 15, 2002 vol. 99 no. 21