How To Choose Your Animal Model

Peter D. Aplan MD

Senior Investigator

Genetics Branch

Outline of today’s talk

• Framework for cancer (leukemia) research.• Approaches to model cancer in mice.• Considerations in choosing a model.

Framework for cancer research

• “Cancer is a genetic disease”– Acquired/inherited– GCR/Single nuc changes

• “Cancer is a developmental disease”– Drosophila developmental mutants (Wnt, Runt, Trithorax, Notch)

• Cancer as an infectious disease– Invasion of normal tissues by rogue cells– Treated with small, cytotoxic molecules (often in combination)– Relapse if not completely eradicated– Important role for the immune system in eradicating rogue cells

“Progress in science depends on new techniques, new discoveries and new ideas, probably in that order.” Sydney Brenner, circa 1980

Koch’s postulates

• The microorganism must be found in abundance in all organisms suffering from the disease.

• The microorganism must be isolated from a diseased organism and grown in pure culture.

• The cultured microorganism should cause disease when introduced into a healthy organism.

• The microorganism must be re-isolated from the inoculated, diseased experimental host and identified as being identical to the original specific causative agent.

Identify the lesion, isolate the lesion, recapitulate the lesion.

Koch’s postulates• The microorganism must be found in abundance in all organisms

suffering from the disease. • The microorganism must be isolated from a diseased organism and

grown in pure culture. • The cultured microorganism should cause disease when introduced

into a healthy organism. • The microorganism must be re-isolated from the inoculated, diseased

experimental host and identified as being identical to the original specific causative agent.

Koch’s postulates (adapted for cancer)• The (cancer gene) must be found in abundance in all organisms suffering from the (particular subtype of cancer). • The (cancer gene) must be isolated from a diseased organism. • The (cancer gene) should cause disease when introduced into a healthy organism. • The (cancer gene) must be re-isolated from (expressed in) the inoculated, diseased experimental host and identified as being identical to the original specific causative agent.

Koch postulates 1 and 2 –identify and isolate the lesion—Gene Discovery

• 1960s-1990s--- Karyotype• New tools in the 1990s

– Expression arrays– SNP, aCGH arrays– Gene/genome re-sequencing

“Progress in science depends on new techniques, new discoveries and new ideas, probably in that order.” Sydney Brenner, circa 1980

Comment— a corollary of this postulate might suggest that key insights come from studies that are not particularly “hypothesis driven”.

New/revised ideas on clonal evolution and pre-malignant lesions.

Koch Postulate 3 and 4—Recapitulate the disease--Utility of animal models for human malignancies

• In vivo verification of putative disease genes.• Understand disease biology.• Pre-clinical evaluation of therapeutic approaches (impetus for

development of palbociclib –trade name Ibrance-- stemmed from mouse breast cancer caused by Cdk4/CyclinD).

• Understand natural history of disease process—clinical presentation of cancer is often a very late stage of disease evolution. Allows for study of pre-malignant lesions.

• Murine hematopoiesis similar—not identical—to human hematopoiesis.

How To Choose Your Animal Model Animals used as biomedical research models

“all models are approximations”

Species Advantage DisadvantageFlies Small, short generation time Not vertebrates

Fish Short generation time, clear embryos Not mammals

Mice Mammals, relatively small, many genetic “tools” available

Not primate; last common ancestor 100 M yrs ago

Rats Same as mice. Bigger than mice. Fewer genetic “tools”. Bigger than mice.

Dogs Outbred species, useful for BMT Costly, pets, ethical issues.

Non-human primates

Similar to humans. Similar to humans.

“2015 Mouse 101 Course”

Classification of murine cancer models

• Spontaneous• Chemically induced• Viral induced• Genetically engineered• Xenotransplant• Tissue transplant (mostly hematopoietic models)

Spontaneous

• Most spontaneous cancers rare in outbred mice.• Can be age-related (hepatoma, lung adenoma).• Investigators noted that certain strains of inbred mice were

pre-disposed to develop cancer (skin, breast, leukemia).• Why?—answers led to seminal advances in understanding

cancer biology.– Vertical transmission and viral etiology.– Concept and cloning of modifier loci (ex. Mom1)

Chemically induced

• Random mutagenesis:– Use low dose mutagen – ie, ethylnitrosourea (ENU) – Can obtain germline (mutagenize sperm) or acquired mutants– Study phenotype of homozygotes, heterozygotes– Large scale projects. Lots of mouse cages, genotyping– Cloning involved loci is laborious, many backcrosses, need genetic

maps.– Demonstrated utility:

• L1210, P388 leukemias (induced by 3-methylcholantrene) used for drug screening and modeling in vitro

• Min (multiple intestinal neoplasia) mutant caused by Apc truncation; led to cloning of Apc gene and critical insights into colon cancer and Familial Adenomatous Polyposis

• Skin cancers and coal/cigarette tar

Viral induced cancer

• MMTV (mouse mammary tumor virus)• MULV (murine leukemia virus)• Vertically transmitted disease (mother’s milk)• Retroviral Insertional Mutagenesis

Retrovirus integration into the genome can transform cells by either oncogene activation or tumor suppressor gene inactivation

Proto-oncogene

OncogeneVirus

Tumor suppressor

Tumor suppressor

Gene activation Viral promoter insertion and enhancer activation

Gene inactivation

Virus

Retroviral Insertional Mutagenesis

Retroviral Insertional Mutagenesis• Retroviral insertion is random at first approximation • Transformation leads to a growth advantage and

clonal expansion which results in tumor formation

• Proviral Integration serves as a tag by which nearby genes are identified through ligation-mediated PCR.

• Multiple viral integrations occur in each mouse

• Common integration sites (CIS) identify nearby candidate genes

Retroviral Insertional Mutagenesis

• Utility:– Discovery of Hoxa9, Meis1, Evi1, Etv6 role in

leukemia– Discovery of Wnt-1 (β-catenin pathway),

Fgf3/4/8, Notch-4 role in breast cancer– Limited by cell type tropism– Extension to most cell types through Sleeping

Beauty Transposon

Transplantation of modified tissue

• Primarily used to study hematopoietic malignancy• Transduce (WT or modified) HSC with viral vector

carrying gene of interest• (Select transduced cells)• Transplant into lethally irradiated recipients• Utility:

– Relatively quick experiments– Can test several gene variants (mutants) simultaneously– Can test combinations of genes– Irradiation of recipients, integration sites, and in vitro selection

can be confounding variables

Xeno (foreign)-grafts• Typically human cancer cells engrafted into

immuno-deficient mice.• Problems with immune rejection,

microenvironment, cytokine/hormones.• Mice lack intact immune system; therefore difficult

to model immunotherapies.• Some investigators have suggested these to be

“animal culture”, one step from tissue culture.• https://www.jax.org/news-and-insights/2006/marc

h/choosing-an-immunodeficient-mouse-model Google “jackson labs immunodeficient”

Xeno-grafts

• Nude (nu/nu) mice.• Homozygous for a mutant Foxn1 gene.• Defective thymic epithelial cells, lack normal

numbers of functional T/B cells.• Graft rejection due to residual innate lymphoid

cells (NK cells).• Hairless, easy to visualize/measure

subcutaneous tumors.• Engraft leukemia poorly.

• Rag1 or Rag2 KO.• Deficient in VDJ recombination; therefore no

mature T/B cells.• Still make NK cells.• Difficult to breed.

Xeno-grafts

• Scid/Scid – spontaneous recessive mutant involving the Pkrdc gene.

• Pkrdc gene required for non homologous end joining (NHEJ; major repair pathway for DNA double strand breaks).

• NHEJ needed for normal VDJ recombination; no VDJ recombination, no T/B cells.

• Leaky. Low, but finite ability (0.1% normal) to produce functional VDJ coding joints.

• Leakiness and spontaneous lymphoma increase with age.• Used extensively for AML engraftment, concept of cancer stem

cells (SLIC, aka Scid Leukemia Initiating Cells) originated from these experiments.

Xeno-grafts

• “Improved” Scid models.• Non-obese diabetic (NOD)/Scid. • NOD developed to model IDDM; immunodeficiency

incompletely characterized, polygenic. • Defective NK, complement, IL-1 (macrophage activator).• NOG/Scid—NOD/IL2rg deficient/Scid. IL2rg (“common

gamma chain”) forms IL2/4/7/15/21 receptor.• Less leaky, less spontaneous lymphoma than Scid.

Xeno-grafts

Xeno-grafts

MISTRG mice—RAG KO, IL2RG KO, express 5 human cytokinesSeem to be useful for engrafting human heme malignancies

Xeno-grafts

• Historically, malignant cell lines used for xenograft.• Recent interest in Patient Derived Xenografts (PDX,

Avatars).• Xenograft from patient primary tumor, usually into Scid or

NOD/Scid mice.• No passage/selection in vitro.• Typically used to predict patient drug response in vivo.• Attractive concept, several commercial entities ($5-50K).• Unproven; mice without immune systems.• Clinical trials in progress.• Perspective: Nature Reviews Cancer 15, 311–316 (2015)

Genetically Engineered Mice (GEM)

• Altered mouse germline; mutations are identical and transmitted to offspring.

• Transgenic– Tissue specific– Conditional (w/r/to time, tissue)

• Gene targeted– “Knock out” KO—gene deletion/inactivation– “Knock in” KI—gene insertion at specific target

locus

Transgenic

FG FG HD

Vav 5’ Vav 3’NUP98-HOXD13 cDNA

• Plasmid construct: Promoter, Gene, Intron, PolyA signal• Promoter: Constitutive, Inducible, Tissue specific• Core Facility; Commercial Facility ($3-20K)

SV40 intron

GH pAsignal

Generation of Transgenic Mice• Generate vector, purify insert.• Micro-inject fertilized ovum (50-100).• Implant into pseudopregnant females.• Tail biopsy to assess integration of transgene (Southern or

PCR).• Typical (good) results– 5-10% of ~80 pups are transgenic.• If none positive: bad DNA, bad embryos, bad injections, bad

luck, lethal transgene (may need to euthanize prior to birth).• Time (optimistic) – 3 months

Evaluation of Transgenic Mice

• Import potential founders• Breed potential founders (How many? Which ones?)• If offspring all negative: bad luck (chimeric founder),

mis-genotyped, germ cell/embryonic lethality.• Transmission rate should be >75%.• Euthanize F1 positive mouse, collect tissues, assess

RNA expression.• Sequence RNA to verify no mutations.• Want >1 founder, decrease possibility that phenotype is

due to integration effect. • Time (optimistic) – 3 months

Conditional Transgenic

• Estrogen Receptor fusion protein (ie, Myc-ER fusion).– Fusion protein in inactive conformation until ligand

(estrogen, Tamoxifen) added.

• Tet-on/off.– Express transgene under control of Tet operon.– Express rTa or tTa under control of tissue specific

promoter.– Turn on or off with Tet(Dox) cycline.

• NB—these are bad for teeth

– Two transgenes.

Gene Targeting (KO/KI)

• Homologous Recombination in ES cells in vitro• Targeting vector construction:

– Targeting arms– Selectable marker– Counter-selection

Germline mNUP98

ApaISstI SstI

pNHKI

ApaI SstI

5’NUP98/HOXD13

Neo

3’NUP98

mNUP98 Exon

hNUP98 Exon

LoxP

pTK-Neo Vector

hHOXD13 ExonmNUP98 Intron

TK

20 Kb 18 Kb

NsiI

NsiI

AmpR

Ori LacZ

Targeting Construct

12

12

Southern ScreenJ1 EF BJ 73 190 191 136

23 kb —

23 kb —

23 kb —

ApaISstI SstI

20 Kb 18 Kb

NsiI

12

SstI

Neo12

SstISstI

Gene Targeting

CoreFacility

PILab

Gene Targeting (KO/KI)

• Chimeric mice (by coat color)• Go germline!! (presume germ cells are also

chimeric)• Screen germline pups (by coat color and DNA).• Stable allele can now be bred.• Realistically, 1 yr timeline.• CRISPR now allows much simpler targeting of

loci in ES cells in vitro

• Cre-Lox system from lambda phage.• Cre recombinase recognizes sequence specific loxP

sites.• Many cell-type specific or inducible Cre transgenic mice

available (ex: CD19-Cre (B cell); LcK-Cre (T cell); Mx1-Cre (inducible)).

Conditional GEM using Cre-Lox

Combinations

• KI/KO with gene transduction/transplantation of HSC.• Transgenic with chemical or retroviral insertional

mutagenesis.

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1 4 7 10 13 16 19 22 25 28 31 34 37 40 43 46 49 52 55 58

Weeks

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urv

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What’s the best model?What’s the question?

• Gene/pathway discovery: Maybe RIM. Have built in “tag” lacking in chemical mutagenesis. But NGS costs suggest re-evaluation.

• Proof of causation: Probably GEM— can start with primary cells. Problem with cell lines is they have many uncharacterized abnormalities. Human IPS cells??

• Disease progression: Probably GEM—watch disease evolve in situ –normal tissue microenvironment.

• Pre-clinical therapeutic: GEM or xenograft.

Pre-clinical therapeuticModel Pros Cons

GEM • Natural microenvironment• Intact immune system• Genetically homogeneous• Transferable and reproducible

• Not human cancer• Not human

pharmacokinetics• May be difficult to generate

large numbers of animals

Xeno • Human cancer• Easy to generate large

numbers of animals• Transferable and reproducible

• Evolution/selection in plastic• Foreign microenvironment• Lack immune system• Not human

pharmacokinetics

PDX • Human cancer• Primary cancer• Easy to generate large

numbers of animals

• Expensive• Not easily transferable• Foreign microenvironment• Lack immune system• Not human

pharmacokinetics

Conclusions

• Clearly delineate your goal (s).

• Read the literature (but not exhaustively or exclusively).

• Consult with senior investigators.

• Begin your studies.

• Iterative cycles of 1-3 are OK.