Kerstin LindbladToh WhiteheadMIT Center for Genome Research Functional

Kerstin Lindblad-Toh Whitehead/MIT Center for Genome Research Functional Genomics Approach Using Mice

advantages of mouse as a model system • The mouse is chosen not only because it is closely related to humans but also because it has more than 100 years of history in genetic analysis • In addition, the mouse is currently the only species for which embryos can be manipulated using available ES-cell technologies • Technologies for freezing embryos and gametes are well established in the mouse, allowing in vitro fertilization to be combined with embryo transfer methods. Thus, valuable mouse lines can be easily and stably maintained in liquid nitrogen for many years while requiring minimal space and manpower.

Some important features that make the mouse such an ideal organism in which to study biological processes relevant to humans include…. its relatively short generation time (10 wk), Its small size, the history of over a century of genetic studies, and the existence of many inbred strains and hundreds of spontaneous mutations. In addition, practical techniques are now available for random mutagenesis and directed engineering of the genome through knockout, knockin, and transgenic techniques. It is thought that mice and humans diverged from a common ancestor 65– 75 million years ago, yet most salient aspects of mammalian physiology have not diverged significantly in these lineages during this time.

Mice are close to humans Humans and mice have 30, 000 genes each, although the mouse genome is 14% smaller than the human genome (2. 5 vs. 2. 9 gigabytes). There is a remarkable degree of synteny between the two genomes: over 90% of the mouse and human genomes can be partitioned into regions of conserved synteny in which the gene order on the chromosome is conserved. Of the 30, 000 protein-coding genes in the mouse genome, 99% of these have a sequence match in the human genome; when considering the entire mouse and human genomes at the nucleotide level, there is 40% identity.

Mouse sequence reveals great similarity with the human genome Extremely high conservation: 560, 000 “anchors” Mouse-Human Comparison both genomes 2. 5 -3 billion bp long > 99% of genes have homologs > 95% of genome “syntenic”

Mouse sequence reveals great similarity with the human genome Extremely high conservation: 560, 000 “anchors” Anchors (hundreds of bases with >90% identity) represent areas of evolutionary selection… …but only 30 -40% of the highly conserved segments correspond to exons of genes!!!

Construction of human–mouse homology clone map Alignment between part of human chromosome 6 (Hsa 6) and mouse chromosome 4 (Mmu 4). A 1. 6 -Mb interval is enlarged, showing part of Hsa 6 q 16. 1 aligned to a 1. 3 -Mb mouse BAC contig.

Mouse: Animal Models of Human Disease Cancer Hypertension Heart disease Diabetes Parkinson’s disease Alzheimers’ disease Huntington disease Tay Sachs Osteogenesis imperfecta Muscular dystrophy Amyotrophic lateral sclerosis Infertility Sickle cell anemia Alpha-thalassemia Beta-thalassemia Cystic fibrosis SCID Familial hypercholesterolemia Growth hormone deficiency Phenylketonuria Atherosclerosis Asthma Alcohol preference Depression

More details about the mouse physical map • found 51, 486 homologous crosslinks btw two genomes • Of the clones in the human genome tile path, 88% are collinear with the mouse BAC map. For individual human chromosomes, coverage by aligned mouse contigs exceeds 80% on all except chromosome 19 (61%) and the Y chromosome (0%). • Of the total coverage of the mouse BAC map (in 211 contigs), 97% (2, 658 Mb) is aligned to the human genome sequence. • Most mouse BAC contigs contained multiple mouse markers (average 57 markers per contig). • coverage of the mouse genome (2. 8 Gb) in mapped BACs is virtually complete: 296 contigs of average size 9. 3 Mb cover an estimated 2, 739 Mb. (~98%) • 275 gaps due to breaks in synteny btw the two genomes.

Mouse ENU mutagenesis • N-ethyl-N-nitrosourea (ENU) • Very high mutation rate • ENU generates point mutations – 44% A/T > T/A – 38% A/T > G/C • Many types of mutations possible, as well as null – Loss-of-function, gain-of-function

Full genome mutagenesis using ENU • ENU is a highly, efficient mutagen – Especially on sperm, also ES cells • Treatment of one animal generates 100 mutations • Screen 300 -500 mouse lines to test for new mutations in every gene • Mapping the mutation is the most difficult aspect

DNA Lesions after ENU treatment Mutation E. coli Drosophila A. T - T. A 1% A. T - G. C - A. T G. C - C. G A. T - C. G G. C - T. A Other 13% 18% 74% 1% 3% Big Blue Mouse mouse germline 33% 13% 67% 3% 3% 3% 0% 41% 7% 38% 3% 3% 11% 5% 42% 7% 5% 2% 2% 1% In Mouse: 85% A. T, 14% G. C, 1% small deletions

Mouse ENU mutagenesis: Mating scheme of ENU-mutagenesis protocols in the mouse ENU induces roughly 1000 –fold more recessive mutations than dominant ones

Dominant Genome Wide ENU * G 0 x * Inv G 1 * G 2 * Targeted Recessive Inversions x * Recessive ENU Genome * G 0 Wide * G 1 EMS * G 3 x * test lethal x * x ENU * G 0 * * G 1 * x * test G 3 x Del G 2 * * carrier * * * carrier Targeted Recessive Deletions * G 2 * Brown & Balling 2001 Curr. Op. Genet. Develop. 11: 268 uninformative

ENU Mutagenesis Modifier Screens ENU Screens in sensitised pathways X [carrying mutations in pathway of interest] F 1 progeny SCREENS

ENU Mutagenesis Modifier Screens - Dominant Enhancers ENU * G 0 r r x r * New dominant mutations Dominant Modifiers New Alleles G 1 * r Progeny test

F 1 ENU mutants with visible phenotypes (a) Nanomouse (b) dominant spotting (c) microphthalmia mutant (d, e) Batface

Transgenic mice Generally, transgenic mice are generated by microinjecting the DNA fragments into the pronuclei of fertilized eggs. This DNA fragment contains new genetic information and is integrated into the genomic DNA, which is transmitted to the next generation.

Random Integration: - DNA incorporated anywhere in genome (not targeted) - copy number can be very high - disrupts the endogenous DNA at insertion site

Random Integration a b x c y ( a b x y )n c

Transgenic insertion as a mutagen Transgenic insertions occur at random with insertional mutation frequencies of 5% to 10%. The mutated genes can be isolated using the inserted transgene as a probe. In transgenic mice overexpressing mouse. TERT, the homozygous offspring from one line showed severe ataxic phenotype. Positional cloning revealed that this transgenic insertion occurred in the intron 1 of the. Unc 5 h 3 gene, which resulted in transcriptional inactivation of this gene.

The first transgenic mice were produced with the transgene, composed of mouse metallothionein-I gene promoter and the structural gene of rat growth hormone (Palmiter et al. , 1982). This transgene confers on mice with a new potential to manifest gigantism when fed with extra zinc, thereby showing the functional transmission of new genetic information.

Specific targeting of the transgene expression Transgenic mice allowed the researchers to monitor the transgene expression in all cell lineages, at any desired stage of development and in postnatal animals, thereby providing new tools to investigate the cis-elements and promoters that are involved in transcriptional regulation. For example, in transgenic mice expressing the human growth hormone (h. GH) under the regulation of the elastase I promoter, the h. GH expression was directed to the pancreatic acinar cells, which exactly corresponds to that of the endogenous elastase I Gene.

Transgenic mouse in studying pathophysiology Disease related phenotypes could be reconstituted in mice through the expression of the pathogenic mutant alleles of a gene. Mutations in specific glycine residues of the pro-alpha 1 collagen gene are associated with the inherited human disease, osteogenesis imperfecta type II. In transgenic mice expressing the mutant pro-alpha 1 (I) collagen gene with substitutions in a glycine residue, a dominant lethal phenotype, which is a characteristic of this human disease, was observed

The transgenic approach is extensively used both to establish heritable tumors in vivo and to reveal the oncogenic potential of a gene. The c-myc oncogene driven by immunoglobulin enhancers leads to lymphoid malignancies in transgenic mice within a few months of their birth In addition to the specific expression of a pathogenic transgene, transgenic technology is utilized to examine the biological functions of the specific cell lineage by eliminating them in vivo. This is accomplished by targeting the expression of a toxic gene, such as Diphtheria toxin, in specific cell lineages. The Elastase I promoter is known to restrict thetransgene expression in pancreatic acinar cells in mice. The transgenic expression of the Diphtheria toxin (DT) A gene under the control of the elastase I promoter/enhancer specifically eliminates pancreatic acinar cells, thereby resulting in mice that lack a normal pancreas (Palmiter et al. , 1987).

Disruption of endogenous gene function: Dominant negative mutations are usually carried out for the elimination of the activities of the wild-type gene products. The expression of dominantnegative mutant genes will compete with and eventually disrupt the endogenous gene functions. This method is specially useful when the in vivo functions of a gene cannot be observed through classical gene targeting for it causes several obstacles such as early embryonic lethality homeostasis and functional redundancy. For example, ASC-2, a recently isolated transcriptional co-activator, when eliminated through gene targeting, induced embryonic lethality, which prevented scientists from analyzing the role of ASC-2 in the postnatal stages). On the other hand, the transgenic mouse overexpressing the dominantnegative mutant of the ASC-2 gene displayed multiple mutant phenotypes in various organs, including eye defects. In this case, the genetic leakiness of the dominant-negative approach was rather beneficial to avoiding the embryonic lethality.

Generation of transgenic RNAi mice with pronuclear injection The transgenic construct containing sh. RNA expression cassette driven by H 1 promoter and a EGFP marker driven by PGK promoter (phosphoglycerate kinase 1 promoter) is linearized and injected into the pronuclei of mouse one-cell embryos. The resultant transgenic mice are marked by the expression of EGFP. The knockdown effect of the target gene can be analyzed directly in F 0 EGFP-positive embryos, or trans-genic RNAi lines can be established first.

Embryonic Stem (ES) Cells - isolated from a pre-implantation embryos - from inner cell mass of blastula stage embryo - cells are undifferentiated - cells are pluripotent - able to differentiate into many (all? ) different cell types in embryo - most importantly germ cells - grown in culture - need cells to divide but not differentiate - longer time in culture = more differentation

Production of transgenic mice. Embryonic stem cells from a mouse are cultured and their genome altered by the addition of a cloned gene. These transgenic cells are selected and then injected into the early stages of a host mouse embryo. Here, the transgenic embryonic stem cells integrate with the host’s embryonic stem cells. The embryo is placed into the uterus of a pregnant mouse, where it develops into a chimeric mouse. The chimeric mouse is then crossed with a wild-type mouse. If the donor stem cells have contributed to the germ line, some of the progeny will be heterozygous for the added allele. By mating heterozygotes, a strain of transgenic mice generated that is homozygous for the added allele. The added gene (the transgene) can be from any eukaryotic source.

Gene targeting in mouse The first gene-targeted mouse was generated using hypoxanthine phosphoribosyl transferase(HPRT) as a target via the homologous recombination in ES cells In order to minimize frequent random integrations, and thusincrease the efficiency of gene targeting, a sophisticated selection method, known as “positive and negative selection”, has been introduced: Neo, neomycin phosphotransferase gene as a positive selection marker; TK or DTA, thymidine kinase or Diphtheria Toxin A, respectively, as negative selection markers.

Homologous Recombination a b a' a c b' b d c' c' d' d'

Homologous Recombination: Non-homologous DNA Flanked by Homologous DNA a b a' a' c b' b' d c' c' d' d'

Targeting Construct for Positive/Negative Selection - To make targeting construct: - a positive selectable marker flanked by two “arms” of homologous sequence - a negative selectable marker outside one homologous arm 1 2 3 4 Neomycin. R 1 HSV-TK 3 4

Gene Targeting using Positive/Negative Selection: Deletion Construct: Homologous Recombinant 1 2 3 4 Neomycin. R 1 HSV-TK 3 4 Neomycin. R 1 2 3

Gene Targeting using Positive/Negative Selection: Deletion Construct: Random Integration x y Neomycin. R a ( x HSV-TK d c Neomycin. R a HSV-TK c d )n y

Selection Strategy: - Positive Selection – G 418 - Neomycin Resistance gene - confers resistance to G 418 - G 418 selects for both: - homologous and random integrations - kills cells that have not taken up DNA - Negative Selection - Gancyclovir - Herpes Simplex Virus Thymidine Kinase (HSV-TK) - sensitive to gancyclovir - selects against random integrants

Targeting: - Electroporate ES cells with Targeting Construct - - Select in G 418 and Gancyclovir - enriches for homologous recombination - Pick individual colonies of resistant ES cells (100 s) - Screen for properly targeted cells - PCR and/or Southern blot - Use targeted cells to make a mouse

Gene Targeting using Positive/Negative Selection: Insertion Construct 1 2 3 4 Neomycin. R 3 2 1 2 HSV-TK 3 3 Neomycin. R 4 3 4