Jessica Götzfried Methods Seminar 03.12.14 Transgenic Animals Jessica Götzfried Methods Seminar 03.12.14

Overview Introduction Historical Background Transgenesis – The method for the production of transgenic animals Examples for the use of transgenic animals Ethical concerns

Introduction A transgenic animal carries a foreign gene that has been deliberately inserted into its genome. Official term: GMO’s (Genetically Modified Organism) Transgene – exogenous DNA that is introduced into the animal Transgenesis is the overall process of introducing exogenous DNA stably into the genome of an animal. These foreign genes are inserted into the germ line of the animal, so they can be transmitted to the progeny.

Why do we need transgenic animals? Research Studying gene expression/gene function Establishing models for studying human diseases (Knock in / knock out mice) As bioreactors: Within the field known as pharming, intensive research has been conducted to develop transgenic animals that produce biotherapeutics (vaccines, antibodies, proteins…) Agricultural importance: Disease resistant animals Improvement of Milk yield Wool characteristics Rate of weight gain Egg-laying frequency

Historical background 1966 – First report of microinjection of mouse embryo 1981 – First transgenic mouse (Gordon and Ruddle) – first time the term transgenic occured 1982 - The ‘Supermouse’ produced by Brinster and Palmiter It was created by inserting a human growth hormone gene in mouse genome. The offspring was much larger than the parents. 1983 – Tissue specific gene expression in transgenic mice 1987 – Transgenic pig, sheep, rabbit and fish 1988 – First patented transgenic mouse ‘Oncomouse’ (Leder)

Transgenesis – the production of transgenic animals Step 1 - DNA Preparation Using recombinant DNA methods, build molecules of DNA containing the gene you desire (e.g., the insulin gene) vector DNA to enable the molecules to be inserted into host DNA molecules promoter and enhancer sequences to enable the gene to be expressed by host cells Marker gene Termination Sequence Promotor Transgene (Gene of interest) Marker Gene Termination Sequence Enhancer Step 2 – Introduction of foreign gene into the animal A - Embryonic stem cell method B - Pronuclear microinjection method

A - The Embryonic Stem Cell Method 1. Embryonic stem cells (ES cells) are harvested from the inner cell mass (ICM) of mouse blastocysts. They can be grown in culture and retain their full potential to produce all the cells of the mature animal, including its gametes. 2. Transform ES cells in culture Expose the cultured cells to the DNA so that some will incorporate it. Microinjection Retro virus Electroporation (introducing DNA from one organism into the cell of another by use of an electric pulse) 3. Transgenic stem cells are grown in vitro. 4. Select for successfully transformed cells. 5. Inject these cells into the inner cell mass (ICM) of mouse blastocysts 6. Embryo transfer Prepare a pseudopregnant mouse (by mating a female mouse with a vasectomized male overnight). The stimulus of mating elicits the hormonal changes needed to make her uterus receptive. Transfer the embryos into her uterus. Hope that they implant successfully and develop into healthy pups.

B - The Pronucleus Microinjection Method 1. Collect eggs from a superovulated female animal 2. Firtilize eggs in vitro before the sperm head has become a pronucleus 3. Inject the male pronucleus with your transgene containing solution When the pronuclei have fused to form the diploid zygote nucleus, allow the zygote to divide by mitosis to form a 2-cell embryo. 4. Implant the embryos in a pseudopregnant foster mother

A – The Pronucleus Microinjection Method Advantages: Most common method for creating transgenic mammalian species Foreign DNA integrates into host cell genome at a random location Often multiple copies of the injected DNA are incorporated at one site (concatemers) Disadvantages: Can’t be used for later developmental stages Skill demanding, tedious, labour intensive, time consuming Inefficient: about 5 % of inoculated eggs develop into transgenic animals 66 % of eggs survive the injection procedure 25 % of the implanted eggs develop into pups 25 % of the pups are transgenic from 1000 inoculated fertilized eggs, 30 – 50 transgenic pups are produced Not all transgenic pups will have the appropriate characteristics (Position effect: Transgene might not be expressed because of the site of integration)

Transgenesis – Step 2 A B

Transgenesis Step 3 - Genotyping of offspring Remove a small piece of tissue from the tail and examine its DNA for the desired gene. Transgenic progenies are screened by PCR to examine the site of incorporation of the gene Some transgenes may not be expressed if integrated into a transcriptionally inactive site. No more than 10–20% will have it, and they will be heterozygous for the gene. Step 4 - Establish a homozygous transgenic strain Mate two heterozygous mice and screen their offspring for the 1 in 4 that will be homozygous for the transgene.

Examples for transgenic animals in lung research

Examples for transgenic animals in lung research

Examples – Transgenic Fish Superfish Increased growth and size Growth hormone gene inserted into fertilized egg. Transgenic salmon grows about 10 – 11 times faster than normal fish Glo fish freshwater zebra fish (Danio rerio) Produce by integrating a fluorescent protein gene from jelly fish into embryo of fish

Examples – Transgenic Cattle and Goats Transgenic cows are made to produce proteins lactoferrin and interferons in their milk. Prion free cows resistant to mad cow disease. Goats 2009 the US FDA granted marketing approval for the first drug to be produced in genetically modified livestock. The drug is called ATryn, which is antithrombin protein (plasminogen activator), purified from the milk of genetically modified goats. Goats that produce spider silk etc. in their milk

Examples – Transgenic monkey ANDi ANDi was the first transgenic monkey, born in 2000. “ANDi” stands for “inserted DNA” spelled backwards. An engineered virus was used to insert the harmless gene for green fluorescence protein (GFP) into ANDi’s rhesus genome. ANDi proves that transgenic primates can be created, and can express a foreign gene delivered into their genome.

Examples – Transgenic Rabbit Alba, the EGFP (Enhanced Green Flurescent protein) bunny Created in 2000 as transgenic artwork

Ethical concerns Is it right to produce transgenic animals for: Research? Production of therapeutics? Art? If yes, even though animals are suffering (unwanted disease symptoms)? Are there any environmental risks? Blurring the lines between species by creating transgenic combinations. There may be health risks associated with transgenics. There may be long term effects on the environment when transgenic animals are released into the field. Various bioethicist argue that it is wrong to create animals that would suffer as a result of genetic alteration.

Thank you!