• Biol/Micr 805 4/14/03
• Pearson-White, sp3i@virginia.edu, 2-0756
• Homologous recombination gene replacement in mice
• Cre loxP tissue specific targeting
• Plug and socket

• derived from inner cell mass of blastocysts
• supported on feeder cells
• can reinject into blastocysts
• will rejoin inner cell mass, contribute to mouse embryo, including germ line
• blastocyst injection
  • or aggregation chimeras
• reimplantation into uterus

• also called homologous recombination gene targeting
• makes use of ES cells to select rare homologous recombinants
• isogenic DNA, length of flanking homology important
• positive/negative selection
• important to maintain totipotency of ES cells during selection, so will contribute to (pass through) the germline
• design of targeting construct is important to allow screening for targeting construct insertion at the homologous site

• first gene targeting in 1987
• hprt, Thomas and Capecchi, and Smithies lab, correction of mutation
• second in 1988
• int-2, Mansour, Thomas and Capecchi, introduction of mutation
• ~140 in 1994

• redundancy of function (MyoD, myf5)
• homeobox genes regulate gene expression universally
• mouse ­ human (hprt)
  • >100 examples of mouse model resembling human disease
• developmental defects mimic human pathologies
  • inflammatory bowel disease from IL-2 KO
• variations in genetic background are important (EGF-R)
• which malfunctions are lethal during embryogenesis

• intraembryonic events
  • blastocyst formation
  • gastrulation
  • heart development
• extraembryonic events
  • trophoblast-decidua connection
  • allanto-chorionic connection
  • yolk sac circulation
  • placental function
• others are not required until birth
  • muscular or neural function
  • Skeleton, teeth
  • digestive system

• http://www.biomednet.com/
  • you need to register and get a free membership
  • the knockout database within biomednet:
    • http://biomednet.com/mkmd
• or TBASE (targeted mutation database at Jackson labs)
  • http://tbase.jax.org/docs/tbase.html
• or Jax (Jackson labs)
  • http://jax.org

• knock-in
• tag-and-exchange
• point mutation, remove selectable marker
• targeting transgenes
• tissue-specific knockout
• inducible, tissue-specific

• point mutation, remove selectable marker
• make more subtle mutations
• selectable markers may cause unknown effects

• do two related proteins have different biochemical functions, and fulfill different roles within the same cells?
• or are the proteins biochemically equivalent, but the divergence in temporal expression pattern explains differences in phenotypes between the two knockouts
• test using knock-in, to replace one with another, under developmental control of the one
• en-2 replacing en-1 (en-1^2ki)
  • en-2 viable, small defect
  • en-1 perinatal lethality
  • en-2ki, rescue, appears normal, showing that en-2 can substitute for en-1

• MyoD KO - viable, fertile, normal muscles, impaired muscle regeneration in adult, after wounding or muscular dystrophy
• Myf5 KO - neonatal lethal, skeletal muscle is fine, stunted ribs caused impaired breathing
• Myf6 (MRF4) KO - 3 phenotypes, from viable to neonatal lethal, muscle development OK
• Myogenin KO - neonatal lethal, skeletal muscle reduced and impaired, myogenin required for normal muscle differentiation

• MyoD KO, normal skeletal muscle, elevation and prolongation of myf5 expression
• Myf5 KO, normal skeletal muscle, unexpected stunting of ribs
• double KO, no skeletal muscle
• Double knockouts and even triple knockouts are becoming common

• measure gene expression of endogenous gene in normal chromosomal context
• check for proper expression of knocked-in gene at targeted locus
  • e.g. en-2 expressed as en-1 would be
• can examine expression in chimeras, or heterozygotes, or homozygotes

Cre-lox tissue specific targeting

• germline knockouts sometimes die as homozygotes prior to the phenotype or function one most wants to study
• methods are now emerging to inactivate a gene only in the desired cell type of interest
  

Cre-loxP

• Two mouse lines are required
• 1. Cre recombinase expressing transgenic line
• Expression in the tissue or cell type of interest
• Various different tissues can be tested with each targeted gene
• 2. Homologous recombinant line in which the deletion of interest is flanked by loxP sites

Cre-loxP targeting

Cre-lox continued

• Cre recombinase-expressing mouse transgenic line(s)
• database of consortium lines at: http://www.mshri.ori.ca/develop/nagy/nagy.htm
• penetrance is still a problem
• Flp/FRT is another, similar method, but less widely used

• Since Cre recombination is incompletely penetrant and can vary from cell to cell, it is now a good idea to include a recombination indicator in targeting constructs
• Examples are loxP-stop-loxP-lacZ or loxP-stop-loxP-eGFP

• to eliminate position effect variegation (PEV)
• to get reproducible high level expression, tissue specificity
• goes through ES cells
• plug and socket

• The second targeting step is detected by selecting against the original marker that was inserted in the first step
• There is a high background of loss of marker gene function in nonrecombinant colonies, for example by spontaneous mutation
• These clones are selected for in culture

• Allows rapid repeated alteration of a chosen target gene

• Uses positive selection in both first and second steps
• Targeting in HPRT-deficient ES cells
• Socket is functional neo gene with a nonfunctional HPRT minigene (ÆHPRT) lacking a promoter and exon 1
• The same socket can be used repeatedly for different plug targeting events

• Plug contains homologous sequences and nonfunctional human HPRT (HPRTÆ) that is complementary to the ÆHPRT in the socket and contains promoter and exon 1
• Recombination between HPRTÆ and ÆHPRT reconstitutes a functional HPRT gene
• And introduces desired mutations engineered in target gene

from Detloff, P. J., J. Lewis, S. W. John, W. R. Shehee, R. Langenbach, N. Maeda and O. Smithies 1994. Deletion and replacement of the mouse adult beta-globin genes by a "plug and socket" repeated targeting strategy. Mol Cell Biol. 14:6936-6943

Plug and socket technology can be combined with Cre/loxP technology

Inducible gene targeting

• tetracycline repressible system (Tet-off)
• tetracycline inducible system (Tet-on)
• ecdysone-inducible system, trinary
• lac repressor

Lac repressor system for inducible gene expression in mice

Recombination indicators