Course objectives The main objective is to develop the ability to critically analyse and interpret the results of the scientific literature and to be able to apply this knowledge to afford new scientific questions. Student Learning Outcomes (SLOS) - Advanced Cell Biology Extending and applying knowledge of cell biology to new situations, interpreting and analysing information Selecting information from a variety of sources Drawing valid conclusions and giving explanations supported by evidence/justification Drawing on knowledge and understanding of cell biology to make accurate statements, describe complex information and integrate knowledge Critically evaluating experimental procedures Communicating cell biology findings/information fully and effectively Analysing and evaluating scientific publications and media reports
TRANSGENIC ANIMALS -animals which have been genetically modified by the artificial introduction of genetic material in every cell ( genetically modified organisms/gmo ) Only for teaching purposes - not for reproduction or sale
Only for teaching purposes - not for reproduction or sale GENETIC MODIFICATION of CELLS - transient vs stable transfection of cells TRANSGENIC ANIMALS - Two methods to produce transgenic animals: 1- DNA microinjection - random insertion 2- embryonic stem cell-mediated gene transfer - random insertion - homologous recombination (double selection) - Rosa26 locus - knock-out animals - knock-in animals - conditional knock-out (cre-lox technique, inducible systems) - genomic analysis to identify genetically modified animals
Only for teaching purposes - not for reproduction or sale GENETIC MODIFICATION of CELLS - transient vs stable transfection of cells TRANSGENIC ANIMALS - Two methods to produce transgenic animals: 1- DNA microinjection - random insertion 2- embryonic stem cell-mediated gene transfer - random insertion - homologous recombination (double selection) - Rosa26 locus - knock-out animals - knock-in animals - conditional knock-out (cre-lox technique, inducible systems) - genomic analysis to identify genetically modified animals
Which vectors can be used for protein expression in eukaryotic transfected cells? Only for teaching purposes - not for reproduction or sale
TRANSIENT TRANSFECTION of CELLS Only for teaching purposes - not for reproduction or sale
Only for teaching purposes - not for reproduction or sale TRANSFECTION METHODS chemical reagents - DEAE-dextran, calcium phosphate co-precipitation lipofection electroporation virus infection
TRANSIENT TRANSFECTION of CELLS Only for teaching purposes - not for reproduction or sale
STABLE TRANSFECTION of CELLS Only for teaching purposes - not for reproduction or sale
STABLE TRANSFECTION of CELLS Only for teaching purposes - not for reproduction or sale when drug selection is used, cells are maintained in nonselective medium for 1 2 days post-transfection, then replated in selective medium containing the drug the use of selective medium is continued for 2 3 weeks, with frequent changes of medium to eliminate dead cells and debris, until distinct colonies can be visualized individual colonies can be isolated by cloning cylinders, selected and transferred to multiwell plates for further propagation in the presence of selective medium individual cells that survive the drug treatment expand into clonal groups that can be individually propagated and characterized
pirespuro2 contains the internal ribosome entry site (IRES) of the encephalomyocarditis virus (ECMV), which permits the translation of two open reading frames from one messenger RNA. After selection with puromycin, nearly all surviving colonies will stably express the gene of interest, thus decreasing the need to screen large numbers of colonies to find functional clones. The expression cassette of pirespuro2 contains the human cytomegalovirus (CMV) major immediate early promoter/enhancer followed by a multiple cloning site (MCS) that precedes stop codons in all three reading frames, a synthetic intron known to enhance the stability of the mrna, the ECMV IRES followed by the gene encoding puromycin resistance (puromycin-n-acetyl-transferase, and the polyadenylation signal of the bovine growth hormone.
STABLE TRANSFECTION of CELLS Only for teaching purposes - not for reproduction or sale
Only for teaching purposes - not for reproduction or sale TRANSGENIC ANIMALS - What is a transgenic animal? - Which kind of genes would you like to over-express? - Which kind of construct do you have to prepare? - How do you insert the construct in the animal? - How do you identify the transgenic animals?
What is a transgenic animal? a transgenic animal defines an animal which has been genetically modified by the artificial introduction of genetic material in every cell the insertion of the foreign DNA (exogenous gene or transgene ) can - confer a gain of function (production of a new protein) - over-expression of an endogenous gene - expression of a mutated form of an endogenous gene - expression of a sirna switching off the expression of a gene -.. Only for teaching purposes - not for reproduction or sale
Which genes would you like to over-express? - It depends from which is your scientific question Which kind of construct do you have to prepare if you want to study: the over-expression of a protein? (ubiquitous / tissue specific / inducible expression) the regulation of a promoter? the down-regulation of a protein? the localization of a protein?..? How can you discriminate between endogenous and exogenous proteins? How can you study the expression of the exogenous protein without the expression of the endogenous protein? Only for teaching purposes - not for reproduction or sale
Only for teaching purposes - not for reproduction or sale Which kind of construct do you have to prepare if you want to study: the over-expression of a protein? (ubiquitous / tissue specific / inducible expression) the regulation of a promoter? the down-regulation of a protein? the localization of a protein?..? How can you discriminate between endogenous and exogenous proteins? How can you study the expression of the exogenous protein without the expression of the endogenous protein?
Only for teaching purposes - not for reproduction or sale TRANSGENIC ANIMALS -transient transfection of cells -stable transfection of cells - Two methods to produce transgenic animals: 1- DNA microinjection - random insertion 2- embryonic stem cell-mediated gene transfer - random insertion - homologous recombination (double selection) - Rosa26 locus - knock-out animals - knock-in animals - conditional knock-out (cre-lox technique, inducible systems) - genomic analysis to identify genetically modified animals
Two methods to produce transgenic mice are widely used: 1 - injecting the construct into the pronucleus of a fertilized mouse egg 2 stably transfecting embryonic stem cells (ES cells) with the construct
1 - injecting the construct into the pronucleus of a fertilized mouse egg Only for teaching purposes - not for reproduction or sale the basic technical approach used for the generation of transgenic animals is called "pronuclear injection : harvest freshly fertilized mouse eggs inject the male pronucleus with DNA https://www.youtube.com/watch?v=xhvyicrm6fq
Only for teaching purposes - not for reproduction or sale
1 - injecting the construct into the pronucleus of a fertilized mouse egg Only for teaching purposes - not for reproduction or sale harvest freshly fertilized mouse eggs inject the male pronucleus with your DNA when the pronuclei have fused to form the diploid zygote nucleus, allow the zygote to divide by mitosis to form a 2-cell embryo implant the embryos into the uterus of a pseudopregnant foster mother
1 - injecting the construct into the pronucleus of a fertilized mouse egg Only for teaching purposes - not for reproduction or sale implant the embryos into the uterus of a pseudopregnant foster mother A pseudopregnant foster mother is a female mated with a vasectomized male: the stimulus of mating elicits the hormonal changes needed to make her uterus receptive. some will implant successfully and develop into healthy pups (usually no more than onethird will)
1 - injecting the construct into the pronucleus of a fertilized mouse egg Only for teaching purposes - not for reproduction or sale DNA integrates at random positions in the genome a loss of function may occur if the transgene interrupts another gene which cells or tissues express the exogenous protein?
Only for teaching purposes - not for reproduction or sale Microinjection in fertilized eggs DNA The transgene is injected into the male pronucleus of a fertilized egg The DNA is inserted in the genome RANDOMLY by non-homologous recombination G0 offsprings from surrogate mothers contain transgene in ALL cells G0 crossed with non-transgenics. Offsprings called FOUNDERS
2- embryonic stem cell-mediated gene transfer During development, there is a stage (the blastocyst) when only two cell types are present: 1-the outer trophoblast cells, which will form the fetal portion of the placenta 2-the inner cell mass, whose cells will give rise to the embryo itself. if an inner cell blast blastomere from one mouse is transferred into the blastocyst of a second mouse, donor cells can contribute to every organ of the host embryo. Inner cell mass blastomeres can be isolated from an embryo and be grown in in vitro culture. These cells are called embryonic stem cells (ES cells). They retain their full potential to produce all the cells of the mature animal, including its gametes. it is not necessary to obtain ES cells from the inner cell mass every time, because ES cell lines are available.
2- embryonic stem cell-mediated gene transfer ES cells are almost totipotent, since each of them can contribute to all tissues, except the trophoblast, if injected into a host embryo. stably transfect ES cells with the construct (using positive and negative selection as discussed later). microinject stably transfected ES cell into another host embryo; ES will integrate into the host. implant the embryos into the uterus of a pseudopregnant foster mother. The result is a chimeric mouse: some of the chimera cells will be derived from the host inner cell mass, some will be derived from the donor stably transfected embryonic stem cells.
2- embryonic stem cell-mediated gene transfer https://www.youtube.com/watch?v=yfm0c4eewqg
2- embryonic stem cell-mediated gene transfer If the treated cells become part of the germ line of the mouse, some of its gametes will be derived from the donor cell. If the chimeric mouse is mated with a wildtype mouse, some of its progeny will carry one copy of the inserted gene. When these heterozygous transgenic mice are mated to one another, about 25% of the resulting offspring will be homozygous for the inserted gene in every cell of their bodies (:the inserted gene will be present in both chromosomes).