TRANSGENIC ANIMALS. transient. stable. - Two methods to produce transgenic animals:

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1 Only for teaching purposes - not for reproduction or sale CELL TRANSFECTION transient stable TRANSGENIC ANIMALS - Two methods to produce transgenic animals: 1- DNA microinjection 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

2 1 - injecting the construct into the pronucleus of a fertilized mouse egg 2- embryonic stem cell-mediated gene transfer

3 RANDOM VERSUS TARGETED GENE INSERTION The basic vectors used for gene insertion place the transgene (from 1 to 200 copies of it) anywhere in the genome. DNA integrates at random positions in the genome - a loss of function may occur if the transgene interrupts another gene - no expression may occur if the trangene is inserted in a silenced DNA region - tissue specific expression may occur if the transgene is inserted downstream a tissue specific promoter - over-expression may occur if many copies of the construct are inserted in the genome If you want to obtain: selective inactivation of a specific gene (KNOCK-OUT) replacement of a gene with a mutated/modified copy of the same gene (KNOCK-IN) proper expression of your transgene (without loss of function of other genes) you need HOMOLOGOUS RECOMBINATION

4 Only for teaching purposes - not for reproduction or sale To obtain: KNOCK-OUT (selective inactivation of a specific gene ) KNOCK-IN (replacement of a gene with a mutated or modified copy of the same gene ) PROPER EXPRESSION of your transgene (without loss of function of other genes) it is necessary: -1- to prepare the construct for homologous recombination -2- to transfer it into cells (fertilized egg or embryonic stem cells?) -3- to select cells in which homologous recombination occurred -4- to transfer them into the animal -5- to verify that offspring contains the transgene - what is homologous recombination?

5 HOMOLOGOUS RECOMBINATION Homologous recombination is a genetic recombination in which nucleotide sequences are exchanged between two similar or identical DNA molecules. Cells use homologous recombination to accurately repair harmful breaks that occur on both strands of DNA. Homologous recombination produces new combinations of DNA sequences during meiosis, the process by which eukaryotes make gamete cells, like sperm and egg cells in animals.

6 MEYOSIS I MEYOSIS II

7 HOMOLOGOUS RECOMBINATION Single crossover Double crossover

8 Flanking region1 Flanking region 2 Homologous recombination Random integration - Which strategy can you follow to obtain homologous recombination and distinguish it from random integration?

9 Homologous recombination Flanking region1 Flanking region 2 Adding to the construct the Herpes Simplex Virus Timidine kinase (HSV-tk) or Diphtheria toxin A- subunit (DTA) Random integration Neo R+ cells Neo R+ /TK + cells HSV-tk will convert gancyclovir into a toxic drug and kills HSV-tk+ cells

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11 (neomycin)

12 In which cells do we have to introduce this construct to obtain homologous recombination? DNA microinjection in fertilized eggs or transfection of embryonic stem cells?

13 Only for teaching purposes - not for reproduction or sale

14 Only for teaching purposes - not for reproduction or sale

15 Mario Capecchi, Martin Evans and Oliver Smithies were awarded the 2007 Nobel Prize for Physiology or Medicine in recognition of their discovery of how homologous recombination can be used to introduce genetic modifications in mice through embryonic stem cells. Mario Capecchi Martin Evans Oliver Smithies

16 Only for teaching purposes - not for reproduction or sale Random insertion Homologous recombination

17 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

18 The ROSA26 locus isolated in 1991 by Soriano and coworkers in a gene-trap mutagenesis screening performed in mouse embryonic stem (ES) cells high frequency of gene-targeting events observed at this locus in murine ES cells located on mouse chromosome 6, spans around 9 kb and consists of 3 exons the ROSA26 gene generates 3 polyadenylated RNAs of which 2, transcribed from the plus strand, lack an open reading frame. The third consists of an antisense transcript coding for a putative protein of 505 aminoacids the ROSA26 protein function remains to date unknown, as mutant mice are viable and lack obvious signs of illness the ROSA26 protein is ubiquitously expressed in embryonic and adult tissues

19 Targeting strategy to insert single-copy transgenes (Tg) into the ROSA26 locus. exons 1 and 2 of ROSA26 (white rectangles) are indicated. The prosa26-1 is an example of targeting vector used to insert transgenes into intron 1 of the ROSA-26 gene. the basic vector contains 5 kb of ROSA26 genomic sequences to drive homologous recombination and the gene for the Diphtheria toxin A-subunit (DTA) as negative selection marker. the transgene (Tg), in most cases preceded by a splice acceptor (SA) site, is cloned into a unique Xba1 (X) site.

20 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

21 X = XbaI R1 = EcoRI? How do you verify the correct homologous recombination?

22 How do you verify the correct homologous recombination? Southern blot

23 How do you verify the correct homologous recombination? Which is molecular weight in the wild type or transgenic mice? X = XbaI R1 = EcoRI

24 How do you verify the correct homologous recombination? Which is molecular weight in the wild type or transgenic mice? X = XbaI R1 = EcoRI DNA digested with EcoRI (R1) +/+ +/- -/ kb kb

25 Can PCR be used to identify transgenic animals? X = XbaI R1 = EcoRI

26 Can PCR be used to identify transgenic animals? -PCR could be used to identify transgenic animals, but often it is not possible to verify the homologous recombination, because the PCR product could be too long; - Southern blot is necessary to verify homologous recombination. - PCR can be used routinely on the offspring of transgenic animals, to identify transgenic animals

27 Once you know you have the transgenic mice, how do you verify the correct expression of the exogenous protein?

28 - Western blot - RT-PCR - immunohistochemistry - If the expression of the transgene is driven by a tissue specific promoter, it is necessary to verify the tissue specific expression - Are your mice viable and fertile? - Is over-expression of the transgene inhibiting the growth and/or the morphogenesis of some organs? Or of the organ where you expressed the transgene? - Is the alteration of a certain organ due to altered apoptosis or proliferation? - Is organ differentiation impaired? - Is gene expression changed?

29 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 lack of a protein? (ubiquitous / tissue specific / inducible/constitutive) the localization of a protein?..? How can you discriminate between endogenous and exogenous proteins? How can you study the activity of the mutated exogenous protein without the expression of the wild type endogenous protein?

30 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

31 KNOCK-OUT MOUSE The elimination of a gene can be achieved through gene targeting to generate a knock-out mouse. Homologous recombination allows a researcher to completely remove one or more exons from a gene, which results in the production of a truncated protein or, more often, no protein at all. WT locus Promoter ATG Exon Linearized targeting vector NEO TK Modified locus Promoter Homologous recombination Exon 1 1 NEO 3 4 ES cells are resistant to geneticin (G418)

32 Example:

33 GENERATION OF A NULL ALLELE AT THE BMP-7 LOCUS - If you analyse by Southern blot the genomic DNA digested with EcoRI, which bands do you expect for +/+, m/+ and m/m animals?

34 GENERATION OF A NULL ALLELE AT THE BMP-7 LOCUS

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36 GROSS MORPHOLOGICAL ANALISYS OF BMP-7 MUTANT EMBRYOS

37 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 lack of a protein? (ubiquitous / tissue specific / inducible) the localization of a protein?..? How can you discriminate between endogenous and exogenous proteins? How can you study the activity of a mutated exogenous protein WITHOUT the expression of the wild type endogenous protein?

38 Giovanna Gambarotta- Only for teaching purposes. KNOCK-IN MOUSE A gene can be eliminated through gene targeting and substituted by a cdna coding for a mutated form of the gene, to generate a knock-in mouse. Homologous recombination allows to completely remove one or more fundamental exons from a gene, and substitute them with the new cdna. Promoter WT locus ATG Exon STOP Linearized targeting vector *=mutation SA ATG 2 * 3 4 cdna STOP polya NEO R TK Homologous recombination Promoter Modified locus Exon 1 ATG STOP 1 2 * SA 3 4 polya NEO R 3 4 SA? cdna

39 Giovanna Gambarotta- Only for teaching purposes. Promoter Modified locus Exon 1 ATG STOP 1 2 * SA 3 4 poly NEO R GT 3 4 AG cdna SA=splice acceptor A