Researchers created a construct containing the promoter and coding sequence of the klf-2 gene in C. elegans fused to gfp to determine the localization of klf-2 expression. The construct was microinjected into C. elegans and klf-2 was found to express in the intestine. Krüppel-like transcription factors play important roles in regulating fat metabolism and dysregulation is associated with obesity-related diseases. Expression of the klf-2::gfp fusion was observed in the intestine of C. elegans throughout development, suggesting klf-2 plays a central role in the intestine, where fat storage occurs. Understanding klf-2 and other KLF molecular mechanisms could help diagnose and treat obesity

1. .
Fig 1
d
d
Fig. B
Macy Decker, Kaleb Davis and Dr. Christopher W. Brey
C.elegans
Research
Group
Marywood Univ.
Overexpression of klf-2 in the intestine of C. elegans
Marywood University, Department of Science, Scranton PA, 18509
(Mello et al. 1991)
Abstract
Krüppel-like transcription factors (klfs) are one of the most
common transcription factors found in all living organisms.
One of their key roles is in regulating fat metabolism. In
humans, there are 17 klf genes, whereas in Caenorhabditis
elegans there are only 3, making the worm an ideal model
organism to study fat metabolism. In this study we focused
on the specific gene klf-2 since the expression localization of
Klf-1 and klf-3 is already known to occur in the intestine. To
determine the localization of klf-2 expression, we created a
construct pJS1.promklf2 which contained ca. 1000 bp of the
klf-2 promoter and the full coding sequence fused to gfp. This
expression construct was microinjected with pRF4, a marker
which allows for selection based on the worm’s mutant roller
phenotype. Upon screening, klf-2 is found to express in the
intestine of the worm. These results are consistent with
klf-1 and klf-3 expression localization profile. By studying the
expression of klf-2, insight into the role klf-2 plays in fat
metabolism can be applied to better our understanding of
Human metabolic diseases.
C. elegans
► A free-living nematode (i.e., worm)
► 40% of genes are homologous to
humans
► Easy to maintain in the laboratory
► Laboratory adapted for genetic
transformation (i.e., microinjection)
Krüppel-like Transcription Factors
► Most abundant class of transcription
factors found in all living organisms
► Half of all metabolic process in the
human body involve klfs
► Dysregulation of klfs are known to play
a role in obesity related diseases
e.g., type-2 diabetes and heart disease
A translational fusion construct was made that contained the 5’ promoter starting
from ATG and the full coding sequence covering all 4 exons (Fig. 1A). The promoter
region and full coding sequence were PCR amplified and cloned into TOPO-TA prior
to subcloning into C. elegans expression vector pPD95.67 (Addgene) containing gfp
as a reporter marker. The resulting klf2::gfp construct was designated as pJSpromkl2
and sequenced (Fig. 1B).
Fig.1
Expression of klf-2: gfp in C. elegans
The plasmid DNA for injection were prepared using ConcertTM rapid plasmid miniprep
system, and then injected into the gonadal syntium of individual C. elegans adult
hermaphrodites at a concentration of 50ng/μl (Mello et al.1991). A plasmid DNA (pRF4)
containing the dominant selectable marker rol-6 gene which was co-injected (50ng/μl
with the reporter construct. When expressed, the worm continuously rolls over, thereby
providing a visible phenotype for the selection of transgenic worms.
d
performs many important functions including digestion, energy storage and
distribution of nutrients. Importantly, intestine is the major site of fat storage in
the worm, thus setting up the foundation for us to investigate the functions of
klf-2 in lipid formation in this study.Microinjection into the syntial gonad of a
young adult hermaphrodite (Fig 2). The F3
generation worms showing a roller phenotype
were collected to observe for gfp expression.
At least three independent lines were examined
for each reporter construct.
Fig 2
Fig.3 Analysis of the expression and localization of klf-2 in C.
elegans by GFP reporter. Gfp expression was observed in intestinal
cells in young adult hermaphrodite (Fig a,b,c). The Expression of gfp
covered the intestine and mid body and tail region of young adult worm.
Transgenic C. elegans worms were observed and paragraphed
using Axioskop 2 plus fluorescent microscope (Zeiss, Germany) using
appropriate filter sets (magnification X400). Note: a non-transgenic
(no gfp expression) WT worm is shown in Fig. d (Ling et al. 2016).
Acknowledgements
References
Conclusion
Discussion
Mello C, et al. (1991). Efficient gene transfer in C. elegans
extrachromosomal maintenance and integration of transforming
sequences. EMBO J. 10: 3959-3970.
Ling et al. Defective lipid metabolism associated with mutation in klf2 and klf-
3: important roles of essential dietary salts on gene expression and
function in fat storage. (submitted to BBA - Molecular and Cell Biology
of Lipids) (April 2016).
As indicated by gfp green fluorescent the
expression of klf-2 was detected in all four larval
(data not shown) and adult stages. During larval
development, the gfp expression was continuously
observed along the region of intestine during the
developing larva and adult stages (Figure 3 a-c),
suggesting that a central role of klf-2 is located in
intestine. The C. elegans intestine performs
many important functions including digestion,
energy storage and distribution of nutrients.
Importantly, intestine is the major site of fat
storage in the worm, thus setting up the
foundation for us to investigate the functions
of klf-2 in future lipid formation studies.
On the medical relevance further understanding
of the molecular mechanisms underlying the
regulation of lipid metabolism by klf-2 and other
KLFs will shed new light on how to diagnose
and treat obesity, thus eventually preventing
obesity related diabetes, cancers
(e.g. breast cancer), and a large array of
cardiovascular diseases.