This document summarizes a large-scale genetic screening effort to identify genes implicated in vascular biology in mammals. The screening is done by systematically targeting and trapping genes in embryonic stem cells, then analyzing resulting mouse knockouts. Genes causing lethality are further studied using RNAi knockdown and cell cultures to analyze effects on blood vessel formation in vitro and in developing embryos in vivo. Over 4,000 mouse knockouts have been analyzed so far, with about 20% showing reduced viability or embryonic lethality. Several promising gene targets for neovascular diseases have been identified through this screening process.

1. Large Scale Genetic Screen to Identify Genes Implicated in Vascular Biology in Mammals
Dennis S. Rice, Kim Paes, Diane C. Markesich, D. Wade Walke, Brian Zambrowicz
Lexicon Genetics Incorporated, The Woodlands, TX 77381
INTRODUCTION
Genetic inhibition of key molecules, such as VEGF and
Delta-like ligand 4, that promote blood vessel formation
often results in lethality during early embryogenesis. Blood
vessel formation also contributes to neovascular diseases
such as wet age-related macular degeneration and promotes
tumor progression. Using high throughput gene-targeting and
gene-trapping techniques in embryonic stem (ES) cells, we
are systematically identifying genes that result in lethality in
mice. The potential for these genes to contribute to blood
vessel formation and organization is studied in vitro using
RNAi knockdown and cultures of targeted ES cells induced
to form embryoid bodies. Immunohistochemical staining of
developing vasculature in heterozygous or homozygous
embryos reveals in vivo effects of gene inhibition during
blood vessel formation. These genetic screens enable the
identification of molecules implicated in blood vessel
formation and provide new targets for therapeutic
intervention in neovascular diseases.
anti-PKC
CHA DAP ENZ GPR INH KNS MAS MEM MSC NHR
PHD PHO PRT PSC RAP SEC SGT SKL TRA TRF
REDUCED VIABILITY or EMBRYONIC
LETHAL GENES BY PROTEIN CLASS
The Human Genome
•GPCRs
•Kinases
•Ion Channels
•Key Enzymes
•Secreted Proteins
•Receptors
Superior Human
Targets
Comprehensive Screening
of Mammalian Physiology
Knock Out All Druggable Genes
Immunology Metabolism Cancer OphthalmologyNeuroscience
• Over 4,000 knockout
mouse lines analyzed to
date
Cardiology
CELL BASED ASSAYS TO TRIAGE LETHAL
GENES FOR DEVELOPMENTAL BIOLOGY
Plate 1 Plate 2 Plate 3 Plate 4
Mock Scrambled
ILK VEGFR2
• RNAi knockdown is confirmed using gene-specific TaqMan
assays (data not shown).
• Targeted disruption of VEGFR2 and ILK, benchmarks in
this in vitro assay, are embryonic lethal.
0
5
10
15
20
25
30
Mock (n=4)
Scram (n=6)
ILK (n=4)
VEGFR2 (n=4)
TotalTubuleLength(mm)
0
10
20
30
40
50
60
70
80
90
100
110
120
Mock (n=4)
Scram (n=6)
ILK (n=4)
VEGFR2 (n=4)
%viablecellsrelative
tomeanofscramblecontrol
Plate 1 Plate 2 Plate 3 Plate 4
A. HUVEC network formation on Matrigel using RNAi
knockdown of Integrin-linked kinase (ILK) or VEGF
receptor 2 (VEGFR2)
B. Cell Viability Assay (Alamar Blue)
M
ock
(n=4)
Scram
(n=6)
ILK
(n=4)
VEG
FR
2
(n=4)
EN
ZYM
E
(n=6)
EN
ZYM
E
(n=6)
EN
ZYM
E
(n=6)
EN
ZYM
E
(n=6)
EN
ZYM
E
(n=6)
EN
ZYM
E
(n=6)
G
PC
R
(n=6)
G
PC
R
(n=6)
G
PC
R
(n=6)
G
PC
R
(n=6)
G
PC
R
(n=6)
G
PC
R
(n=6)
0
5
10
15
20
25
30
35
40
45
TotalTubuleLength(mm)
NETWORK FORMATION and CELL VIABILITY
FOLLOWING RNAi KNOCKDOWN in HUVECs
• Targeted ES cell clones that produce high percentage
chimeras transmitting coat color but not the targeted
mutation identify potential haploinsufficient lethal or sex-
linked lethal genes.
Wild type ES Cells Enzyme Knockout ES Cells
Anti-CD31 (green) or alpha
smooth muscle actin (red)
• Approximately 20% of knockout lines exhibit
reduced viability or embryonic lethality
• Cell based assays are used to identify the most
promising targets for timed-matings in the
Developmental Biology Program
CONCLUSION
Lexicon is systematically discovering genes critical for vascular
development by using high throughput gene knockouts in mice.
These genetic screens enable the identification of molecules
implicated in blood vessel formation and provide new targets
for therapeutic intervention in neovascular diseases.
EMBRYOID BODIES INDUCED to
UNDERGO SPROUTING ANGIOGENESIS
A. Results from a screen that included 6 different
enzymes and 6 different GPCRs
M
ock
(n=4)
Scram
(n=6)
ILK
(n=4)
VEG
FR
2
(n=4)
EN
Z
(n=6)
EN
Z
(n=6)
EN
Z
(n=6)
EN
Z
(n=6)
EN
Z
(n=6)
EN
Z
(n=6)
G
PR
(n=6)
G
PR
(n=6)
G
PR
(n=6)
G
PR
(n=6)
G
PR
(n=6)
G
PR
(n=6)
0
10
20
30
40
50
60
70
80
90
100
110
120
%viablecellsrelative
tomeanofscramblecontrol
Enzyme GPCR GPCR
B. Cell Viability Assay (Alamar Blue)
Wild type Knockout
E13.5 Embryo Secreted Protein
AS PD3 Sense
PD6 PD6
NFL
CC
Vascular hemorraging
observed in HOM embryo
High expression observed in
developing retinal vasculature