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Laboratory Animal Science (Subject) / Transgenic animals (Lesson)

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• Define TG animal are genetically modified organisms with DNA from another source inserted into their genome --> Foreign genes are inserted into the germ line of the animal, so it can betransmitted to the progeny AIM: produce food/certain proteins
• Distinguish between different forms of TG techniques 1. Introduction of another gene into animal, both genes are active = classic TG mouse 2. KO animal --> in active gene, either conventional KO (whole body KO in each cell at all times) or conditional KO (tissue and time and age specific) 3. Knock-in animal, only the mutated gene is active
• How are classic TG mice generated? 1. Generation of the “Targeting Vector“  -- in vitro, a promotor sequence is chosen and oocytes are produced (mating overnight of 1 female and 1-2 males), which are then isolated (plug check, oocyte isolation)  2. Injection of targeting vector into the pronucleus --> random (often multiple) integration takes place 3. Transfer of the embryo into pseudopregnant female (hormones) 4. Screening of the offspring --> either TG or WT, often by use of GFP, or expression studies (PCR of genomic DNA) 5. Characterization of the transgenic mouse
• How are conventional KO mice generated? by Targeted mutagenesis in embryonic stem cells 1. targeting vector with positive and negative selection markers is created  2) Electroporation and integration into ES cells, which often may be random due to homologoues/non-homologous recombination ---> Positive(e.g. Neomycin)/Negative-(e.g. Ganciclovir) selection markers are used to reduce the number of clones with random integration  --> Cells with integration at the specific site survive, cells with random integration die 3. ES cell colonies are grown and through specific selection of the targeting vector, only the "right" cells survive --> detection via southern blot 4. Identify the right clones and microinjection into blastocysts of pseudopregnant females (chimeras can be selected) Conditional KO
• What are the advantages of conditional KOs? it is time/age (...) specific and inducible --> investigate the function of a molecule in specific cells --> investigate otherwise embryonically lethal genes Options:  Cre/loxP-SystemKnock-inInducible CreDTR-System
• Explain Cre/loxP-System Aim: time-, tissue-specific knockout of a gene by a site specific recombinase -Cre-Recombinase: Causes recombination (Recombinase from Phage P1) which recombines short target sequences which are marked at the start and the end by a loxP sequence (and is under control of tissue specific promotors or an inducible promotor) -Placing of Lox P sequence allows for recombination between two target sequences  -Cre-Recombinase recongnizes the loxP-Sequence, cuts the DNA and deletes the DNA between the loxP sites --> tissue specific and inducible deletion
• Klausurfrage: Difference between “Classic“ Transgene and KO animals? Classic TG Generation by introduction of DNA into the oocyte and random integration- by selecting promoter/gene,  the expression of any gene at any position is possible- Main disadvantage: high variation due to random integration/copy number Knockout animals: targeted mutagenesis with either inactive gene or active mutatated gene - Animals with artificially removed gene segment- Generation by homologous recombination in ES cells- Complex technology --> Knock out (conventional/conditional) -->Knock in (reporter mouse)
• What is CRISPR? Clustered Regular Interspaced short Palindromic Repeats Cas = cripr associated, system of helicases and nucleases natural defense mechanisms of bacteria and archaea -->they use CRISPR-derived RNA and various Cas proteins, including Cas9, to foil attacks by viruses and other foreign bodies  Principle: CRISPR “spacer” DNA sequences are clustered regularly between interspaced short palindromic repeats  while the palindromic repeats are identical, each spacer is unique and matches perfectly with viral/bacteriophage DNA (or any DNA you want,if you engineer the system) the genes of the Cripr associated system are transcribed into helicases and nucleases (cas complex) which are equipped with CRISPR RNA capable of guiding the Cas enzymes  to matching sequences of DNA --> When "target DNA" is found  enzymes produced by the CRISPR system – bind to that DNA and cut it --> unknown DNA will also be incorporated by the CRISPR DNA -->can be done in vitro/in vivo (oocyte) Using modified versions of Cas9, researchers can activate gene expression instead of cutting the DNA. These techniques allow researchers to study the gene’s function.
• define Knock-in one-for-one substitution of DNA sequence information in a genetic locus or the insertion of sequence information not found within the locus -->targeted insertion, e.g. to study the function of a certain promotor

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