Transcript Screening of Evolutionarily Conserved Enhancers in the Embryonic

Screening of Evolutionarily Conserved Enhancers in the Embryonic Spinal Cord
Joe Knoedler, Amgen Scholars Program
Gene Expression Laboratory at the Salk Institute
Primary Investigator: Dr. Samuel Pfaff
Introduction
In eukaryotes, genes can be regulated by non-coding regions termed
‘enhancers’. Enhancers differ from conventional promoters in that they
can be hundreds of thousands of base pairs away from the genes they
regulate. The Pfaff lab’s primary focus is to determine the genetic
programs governing the development of the circuitry of the spinal cord.
Enhancers may play an important role in this event. To this end, my
objective was to investigate the expression pattern of evolutionarily
conserved enhancer regions (ECRs) in the chick spinal cord (Figure
from Pfaff and Goulding 2005).
Experimental
Procedure
Vectors were prepared by cloning
ECRs from human genomic DNA
using PCR. They were then ligated
into backbones containing a CRErecombinase. The constructs were
cultured in E. coli. Chick embryos
were transformed with the construct
and an RFP reporter plasmid via in
ovo electroporation.
Preliminary Investigation
Collaborators in the Pennachio Lab (Lawrence
Berkeley National Laboratory)identified ~1100
noncoding regions conserved in human, chick
and mouse. They then made transgenic mice
with the putative enhancer genes coupled to ßgalactosidase and used whole-mount stating to
get a gross overview of expression patterns. 281
of these were active in the neural tube (Figures
courtesy of William Alaynick).
Results
Section of neural tube of transformed
chicken embryo. Cells that have
taken up the RFP construct fluoresce
red; cells that have taken up both and
activate enhancer region express a
CRE-recombinase that turns RFP into
GFP. Thus, this enhancer may be
involved in differentiation of green
cells.
Acknowledgements
Enhancers in Development
Enhancers can regulate development by regionally
targeting gene expression. Left, a construct with
whole Hb9 promoter targets expression to ventral
neural tube; right, truncation of enhancer drives
nonspecific GFP expression across dorso-ventral
axis (Figure from Lee et al 2004)
Special thanks to: Magda Gramada, director of UCSD Amgen Scholars program, Will
Alaynick, my postdoctoral mentor, Sam Pfaff, my PI, and the Amgen Foundation for
funding my internship.
References
Goulding, M. and Pfaff, S. L. (2005). Development of Circuits that
Generate Simple Rhythmic Behaviors in Vertebrates. Current Opinion
in Neurobiology 15, 14-20.
Lee, S., Jurata, L. W., Funahashi, J., Ruiz, E. C. and Pfaff, S. L.
(2004). Analysis of Embryonic Motoneuron Gene Regulation:
Derepression of General Activators Function in Concert with Enhancer
Factors. Development 131, 3295-3306.