species- Mus musculus Engineering the mouse genome David Ornitz

How do we analyze gene function in mice? Gene addition (transgenic approach) Permits GOF, DN and knockdown experiments Ectopic (spatial or temporal) expression Allows gene regulatory elements to be tested Allows populations of cells to be marked with a reporter gene Targeted mutations Specific genes can be targeted Unexpected phenotypes (lethal phenotype may result prior to the spatial and temporal site of interest) Must be very careful to make a null allele Tissue-specific (conditional) targeted mutations Provides some of the best features of gene targeting and transgenic approaches May be combined with enhancer trap and gene trap experiments. An effective method to circumvent embryonic lethality.

Breeding mice gestation period-19 days (range is 18-21 days depending on strain) age at weaning-21 days sexual maturity-females 4-5 weeks, males-6-8 weeks birthweight-1 gm weaning-8-12 gm adult-30-40 gm

Preimplantation mouse development

Aggregation chimeras Before the use of microinjection aggregation chimeras were the only way to genetically modify cells and test them during mouse development Morula aggregation, used to make chimeras between two different genetic backgrounds ES/EC cell chimera add genetically modified cells to a mouse

Routes for Introducing Genes into Mice 1) Microinjection of DNA into zygotes 2) Injection of embryos with recombinant virus 3) Transfection of ES cells with cloned DNA Selection, Characterization Chimera formation Transgenic Mice

Transgenic Mice: Gene addition Random insertion of DNA into the mouse genome Permits GOF, DN and knockdown experiments Allows gene regulatory elements to be tested Allows populations of cells to be marked with a reporter gene Occasionally allows endogenous genes to be trapped

Components of a Transgene promoter + enhancer gene coding sequence or cdna polyadenylation signal promoter cdna splice/poly A Things that are good: introns Things that are bad: plasmid sequence, lack of introns

example: Elastase Promoter cell-type specific expression 200 bp is sufficient for expression Pr/En hgh poly A Pr/En v-ras splice/poly A cdna

Transgenic mouse issues: Tissue specificity ectopic expression chromosomal integration site may affect expression Temporal specificity Level of expression Insertional mutagenesis

How to make a transgenic mouse 1. Fusion Gene Construct 2. Superovulated Female Promoter ATG Coding Sequence p(a) Fertilized Eggs Microinjection 3. Germline Integration 4. DNA Analysis TRANSGENIC MOUSE 5. Breeding

Manipulating the Mouse Embryo a laboratory manual, CSHL press from

Homologous recombination using embryonic stem cells First completely unbiased experiment of gene function in an entire mammalian organism. Discover unanticipated early embryonic roles Potential problems: Embryonic lethality Redundancy

Events leading to the development of Embryonic Stem Cells Teratoma tumors composed of various tissues foreign to their site of origin. can be formed by transplanting pieces of embryos to extra uterine sites. Teratocarcinoma undifferentiated malignant stem cells, metastasize, lethal made by transplanting day 6-7 mouse embryos under the kidney capsule resulting tumors can be passaged and cultured to yield embryonal carcinoma cells

Embryonic Stem Cells-cont. EC cell lines variety of stages of differentiation and variable capacity to differentiate exponential growth and feeder cells are required to prevent differentiation differentiation can be induced by aggregation differentiation can be induced by drugs, RA or DMSO. ES cells a normal pleuripotent cell line isolated from normal embryo without passing through a tumor stage. when reintroduced into the embryonic environment ES cells can generate high grade chimeras. essential to grow on feeder cells (STO fibroblasts or MEFs). LIF/DIA is required to maintain pleuripotency of ES cells.

Establishment of ES cell lines: transfer intact blastocysts into culture grow to stage of early post implantation embryo dissociate embryonic from extraembryonic tissue continue to culture ICM. 2 days after disaggregation of ICM 4 days after disaggregation First passage

Chimeric mouse ES cells derived from 129/SV strain, agouti coat color injected into a C57/B6 blastocyst. Mate chimeric mouse to Black mouse (C57/B6J) identify agouti offspring

Gene Knockout critical X

Gene Knockout critical genetic engineering using embryonic stem cells X Mario R. Capecchi Sir Martin J. Evans Oliver Smithies The Nobel Prize in Physiology or Medicine 2007 "for their discoveries of principles for introducing specific gene modifications in mice by the use of embryonic stem cells"

Gene Knockout critical genetic engineering using embryonic stem cells X

Practical issues for basic gene targeting: length of homology probes to detect homologous recombination vector design Target gene Targeting vector Targeted allele

Targeted mutations gene addition Target gene Targeting vector Targeted allele random integration

Issues in interpreting targeted mutations Must be very careful to make a null allele haplotype insufficient recessive Prove that an allele is null gene expression protein expression assay for activity of protein Other types of alleles hypomorphic allele dominant negative linked random mutation - generate multiple ES lines recessive

Xu, X., Weinstein, M., Li, C., Naski, M., Cohen, R. I., Ornitz, D. M., Leder, P., and Deng, C. (1998). Fibroblast growth factor receptor 2 (FGFR2)-mediated regulation loop between FGF8 and FGF10 is essential for limb induction, Development 125, 753-765. Arman, E., Haffnerkrausz, R., Chen, Y., Heath, J. K., and Lonai, P. (1998). Targeted disruption of fibroblast growth factor (Fgf) receptor 2 suggests a role for fgf signaling in pregastrulation mammalian development, Proc. Natl. Acad. Sci., U S A 95, 5082-5087.

Issues in interpreting targeted mutations - cont. Neighboring gene effect PGK promoter - neo may influence a nearby gene remove the selection cassette to avoid this potential problem Unexpected phenotype lethal phenotype may result prior to the developmental stage of interest Targeted allele PGK-Neo

Cell, Vol. 85, 1 4, April 5, 1996, Copyright 1996 by Cell Press Know Your Neighbors: Three Phenotypes in Null Mutants of the Myogenic bhlh Gene MRF4 E. N. Olson,* H.-H. Arnold, P. W. J. Rigby, and B. J. Wold c

Removing the Neo selection cassette critical genetic engineering using embryonic stem cells PGK-NEO loxp loxp flox = flanked by lox X PGK-NEO X

Removing the Neo selection cassette critical genetic engineering using embryonic stem cells PGK-NEO loxp loxp flox = flanked by lox X germline promoter - Cre recombinase PGK-NEO X

Removing the Neo selection cassette critical genetic engineering using embryonic stem cells PGK-NEO loxp loxp flox = flanked by lox X germline promoter - Cre recombinase X

Advanced gene targeting issues Targeting one allele versus both alleles Gene replacement using recombinases Knockin mice

Conditional tissue-specific targeted mutations provides some of the best features of gene targeting and transgenic approaches may be combined with enhancer trap and gene trap experiments the targeted gene can be modified using cre and flip recombinases may be used in conjunction with inducible promoters

Regulated activation/inactivation of a gene using CreER fusion proteins critical loxp loxp flox = flanked by lox critical X

Regulated activation/inactivation of a gene using CreER fusion proteins critical loxp loxp flox = flanked by lox tissue specific promoter - CreER recombinase Cytosol critical X

Regulated activation/inactivation of a gene using CreER fusion proteins critical loxp loxp flox = flanked by lox tissue specific promoter - CreER recombinase + tamoxifen nuclear translocation Cytosol critical X

EUCOMM gene targeting vector Frt SA-βgeo-PA LoxP PGK -neo Critical 5' hom ology 3' hom ology

EUCOMM gene targeting vector Frt SA-βgeo-PA LoxP PGK -neo Critical 5' hom ology 3' hom ology Frt SA-βgeo-PA SA-CreER-PA LoxP PGK -neo Critical 5' hom ology 3' hom ology

Mouse Genetic Models Core!!