Making Transgenic Plants and Animals

Making Transgenic Plants and Animals • Why? • Study gene function and regulation • Generate new organismic tools for other fields of research. • Cure genetic diseases. • Improve agriculture and related raw materials. • Generate new systems or sources for bioengineered drugs (e.g., use plants instead of animals or bacteria).

Transgenic Mice The organism of choice for mammalian genetic engineers. - small - hardy - short life cycle - genetics possible - many useful strains and tools

The Nobel Prize in Physiology or Medicine, 2007 Mario R. Capecchi, Martin J. Evans and Oliver Smithies for their discoveries of "principles for introducing specific gene modifications in mice by the use of embryonic stem cells" M. Capecchi Univ. of Utah Sir M. Evans Cardiff Univ., UK O. Smithies UNC Chapel Hill

The Problem with trying to make KOs: Random DNA Integration • DNA can integrate into the genome by homologous (H) or non-homologous (N-H) recombination • Frequency ofN-H>>H (by at least 5000-fold) in mammalian cells • If you want H integrants, which you need for knock-outs, you must have a selection scheme for those.

Vector for integrating a transgene by HR (i.e., into a specific site) tk1 & tk2 - two copies of a Herpes Simplex Virus thymidine kinase gene (makes cells susceptible to gancyclovir) Neo- neomycin resistance gene Homologous regions - homologous to the chromosomal target Transgene - foreign gene

Example of what happens with N-H recombination Transformed cells are neo-resistant, but gancyclovir sensitive. homol-->

What happens with HR If DNA goes in by HR, transformed cells are both neo-resistant and gancyclovir-resistant! Use double-selection to get only those cells with a homologous integration event.

From Fig. 5.40 • To knock-out a gene: • Insert neo gene into the target gene. • Transform KO plasmid into embryonic stem cells. • Perform double-selection to get cells with the homologous integration (neo & gangcyclovir resistant). • Inject cells with the knocked-out gene into a blastocyst. 1. KO KO 2,3.

How to make a transgenic mouse. With DNA (mouse)

Chimeric mouse

(a) If the recipient ES cells are from a brown mouse, and the transformed (transgenic) ES cells are injected into a black (female) mouse, chimeras are easily identified by their Brown/Black phenotype. (b) To obtain a completely transgenic KO mouse (where all cells have a KO gene), mate the chimera with a black mouse. Some of the progeny will be brown (which is dominant), indicating fertilization with a germ-line cell (gamete) that ultimately came from a KO-ES cell. Only about 50% of the brown progeny mice, however, will have the KO allele, because the transgenic ES cell that underwent meiosis to produce the germ-line cell was probably heterozygous for the KOed gene. (c) To obtain a homozygous KO mouse (both alleles are KOs), cross brown heterozygotes, and ~1/4 of the progeny will be homozygous.

Not necessarily 3:1 Similar to Fig. 5.41

Gene therapy in humans presents some formidable problems • If you could introduce the gene in early development (e.g., eggs? or blastocyst) might could cure (or partially cure) many diseases. • How to fix them later, as a child, adolescent, adult, etc.? • Transgenic technology + stem cell technology = many interesting possibilities

Making Transgenic Plants and Animals