Researchers used zinc-finger nucleases (ZFNs) to generate knockout rats by targeting three genes - green fluorescent protein (GFP), Immunoglobulin M (IgM), and Rab38. ZFNs were microinjected into rat embryos to induce mutations in the target genes. Of 295 founder animals screened, 35 (12%) contained targeted mutations, including full knockout of the GFP transgene in some animals. Mutations were transmitted to offspring, demonstrating the ability of ZFNs to disrupt genes and induce heritable mutations in the rat genome. This technique allows for targeted genetic modification of the rat, an important model for studying human disease.
Variable transcriptional adaptation between the laboratory (H37Rv) and clinic...Santhi Devasundaram
The remarkable success of M. tuberculosis as a pathogen is largely due to its ability to
persist within the host for long periods. To develop the effective intervention strategies, understanding the biology
of persistence is highly required. Accumulating evidences showed oxygen deprivation (hypoxia) as a potential
stimulus for triggering the transition of M. tuberculosis to a non-replicating persistent state analogous to
latency in vivo. To date, in vitro hypoxia experimental models used the laboratory adapted isolate H37Rv and
very little is known about the behavior of clinical isolates that are involved during disease outbreaks. Hence,
we compared the transcription profiles of H37Rv and two south Indian clinical isolates (S7 and S10) under hypoxia
to find differences in gene expression pattern.
Variable transcriptional adaptation between the laboratory (H37Rv) and clinic...Santhi Devasundaram
The remarkable success of M. tuberculosis as a pathogen is largely due to its ability to
persist within the host for long periods. To develop the effective intervention strategies, understanding the biology
of persistence is highly required. Accumulating evidences showed oxygen deprivation (hypoxia) as a potential
stimulus for triggering the transition of M. tuberculosis to a non-replicating persistent state analogous to
latency in vivo. To date, in vitro hypoxia experimental models used the laboratory adapted isolate H37Rv and
very little is known about the behavior of clinical isolates that are involved during disease outbreaks. Hence,
we compared the transcription profiles of H37Rv and two south Indian clinical isolates (S7 and S10) under hypoxia
to find differences in gene expression pattern.
My talk to the PhD students NRP at the Doctoral Training Programme Summer Conference 2015, The Assembly House, Norwich, Thursday 18th June.
Notes and acknowledgments at http://kamounlab.tumblr.com/post/121748816600/what-are-world-class-science-outputs
Hematological Parameters of three Strains of Local Cocks in Northern NigeriaIJEAB
The study was conducted to determine the hematological parameters of three strains of the Nigerian indigenous cocks. A total of 15 sexually matured (14-18 month of age) breeders cocks comprising (5 normal feathered, 5 frizzled feathered and 5 naked neck) were used for the experiment. The study was conducted from October to December 2016 at the Teaching and Research Farm University of Maiduguri. Blood samples were collected from 9 breeder’s cocks which were randomly selected 3 per genotype and used for hematological parameters examination. Hematological examination such as Packed Cell Volume (PCV), Red Blood Cell ( RBC) , Haemoglobin (Hb), White Blood Cell (WBC), Mean Corpuscular Haemoglobin concentration ( MCHC), Mean Corpuscular Haemoglobin (MCH) and Mean Corpuscular Volume ( MCV) showed significant (P<0.05)> 0.05) different between normal feathered and frizzle feathered but there is significant difference ( P< 0.05) with naked necked cock. Neutrophil ( N) showed significant (P<0.05)>0.05) difference between normal feathered and naked neck feathered , fizzle feathered and necked neck respectively but showed significant (P<0.05) difference between fizzle feathered and normal feathered respectively for M and E. the study concluded that variation in the heamatoloical parameters between three strains of local chicken in Nigeria is due to difference in their genetic makeup.
JGI: Genome size impacts on plant adaptationjrossibarra
Genome size may impact how plant genomes adapt, offering larger mutational targets leading to more adaptation from standing variation and more adaptation in noncoding regions.
My talk to the PhD students NRP at the Doctoral Training Programme Summer Conference 2015, The Assembly House, Norwich, Thursday 18th June.
Notes and acknowledgments at http://kamounlab.tumblr.com/post/121748816600/what-are-world-class-science-outputs
Hematological Parameters of three Strains of Local Cocks in Northern NigeriaIJEAB
The study was conducted to determine the hematological parameters of three strains of the Nigerian indigenous cocks. A total of 15 sexually matured (14-18 month of age) breeders cocks comprising (5 normal feathered, 5 frizzled feathered and 5 naked neck) were used for the experiment. The study was conducted from October to December 2016 at the Teaching and Research Farm University of Maiduguri. Blood samples were collected from 9 breeder’s cocks which were randomly selected 3 per genotype and used for hematological parameters examination. Hematological examination such as Packed Cell Volume (PCV), Red Blood Cell ( RBC) , Haemoglobin (Hb), White Blood Cell (WBC), Mean Corpuscular Haemoglobin concentration ( MCHC), Mean Corpuscular Haemoglobin (MCH) and Mean Corpuscular Volume ( MCV) showed significant (P<0.05)> 0.05) different between normal feathered and frizzle feathered but there is significant difference ( P< 0.05) with naked necked cock. Neutrophil ( N) showed significant (P<0.05)>0.05) difference between normal feathered and naked neck feathered , fizzle feathered and necked neck respectively but showed significant (P<0.05) difference between fizzle feathered and normal feathered respectively for M and E. the study concluded that variation in the heamatoloical parameters between three strains of local chicken in Nigeria is due to difference in their genetic makeup.
JGI: Genome size impacts on plant adaptationjrossibarra
Genome size may impact how plant genomes adapt, offering larger mutational targets leading to more adaptation from standing variation and more adaptation in noncoding regions.
cloning. Second, it is sensitive. Activities canbe detected WilheminaRossi174
cloning. Second, it is sensitive. Activities can
be detected in the purified GST-ORF pools
that simply cannot be detected in extracts or
cells, the starting point of both conventional
purification and expression cloning. Because
the GST-ORFs are individually expressed at
high levels and are largely free of extract
proteins after purification, activities can be
measured for hours without competing activ-
ities that destroy the substrate, the product, or
the enzymes.
In addition to the conventional use demon-
strated here, this array could be used in two
other ways: (i) to determine the range of poten-
tial substrate proteins for any protein-modifying
enzyme (such as a protein kinase) before genet-
ic or biochemical tests to establish authentic
substrates and (ii) to identify genes encoding
proteins that bind any particular macromole-
cule, ligand, or drug. Thus, one could rapidly
ascribe function to many presently unclassified
yeast proteins, complementing other genomic
approaches to deduce gene function from ex-
pression patterns, mutant phenotypes, localiza-
tion of gene products, and identification of in-
teracting partners.
References and Notes
1. H. Simonsen and H. F. Lodish, Trends Pharmacol. Sci.
15, 437 (1994).
2. Plasmid pYEX 4T-1 (Clontech, Palo Alto, CA) was
modified by the addition of a 140-nucleotide recom-
bination domain, 39 of its Eco RI site, linearized within
the recombination domain by restriction digestion,
and cotransformed with a genomic set of reamplified
ORFs that had the same ends as the linearized plas-
mid [ J. R. Hudson Jr. et al., Genome Res. 7, 1169
(1997)] into strain EJ 758 [MATa his3-D200, leu2-
3,112, ura3-52, pep4::URA3], a derivative of JHRY-
20-2Ca (5). Transformants obtained on synthetic
minimal (SD) 2 Ura drop-out plates [F. Sherman,
Methods Enzymol. 194, 3 (1991)] (.100 in all cases,
and more than five times the cut vector in 97% of the
cases) were eluted in batch and saved in 96-well
microtiter plates. The library contains 6080 ORF-
containing strains and 64 strains with vector only.
3. Cell patches were inoculated in SD 2 Ura liquid
medium, grown overnight, reinoculated, and grown
overnight in SD 2 Ura 2 Leu medium, and then
inoculated into 250 ml of SD 2 Ura 2 Leu medium,
grown to absorbance at 600 nm of 0.8, and induced
with 0.5 mM copper sulfate for 2 hours before har-
vest [I. G. Macreadie, O. Horaitis, A. J. Verkuylen,
K. W. Savin, Gene 104, 107 (1991)]. Cells were re-
suspended in 1 ml of buffer [50 mM tris-HCl (pH 7.5),
1 mM EDTA, 4 mM MgCl2, 5 mM dithiothreitol (DT T),
10% glycerol, and 1 M NaCl] containing leupeptin (2
mg/ml) and pepstatin (1 mg/ml), and extracts were
made with glass beads [S. M. McCraith and E. M.
Phizicky, Mol. Cell. Biol. 10, 1049 (1990)], followed
by supplementation with 1 mM phenylmethylsulfo-
nyl fluoride and centrifugation. GST-ORF fusion pro-
teins were purified by glutathione agarose chroma-
tography in buffer containing 0.5 M NaCl, essentially
as described [ J. ...
Next Generation Sequencing and its Applications in Medical Research - Frances...Sri Ambati
The so-called “next-generation” sequencing (NGS) technologies allows us, in a short time and in parallel, to sequence massive amounts of DNA, overcoming the limitations of the original Sanger sequencing methods used to sequence the first human genome. NGS technologies have had an enormous impact on biomedical research within a short time frame. This talk will give an overview of these applications with specific examples from Mendelian genomics and cancer research. #h2ony
The influence of reduced oxygen availability on gene expression in laboratory...Santhi Devasundaram
Virtually all dormant
models against tuberculosis tested in animals used laboratory strain H37Rv or Erdman strain. But major
outbreaks of tuberculosis (TB) occur with the strains that have widely different genotypes and phenotypes
compared to H37Rv. In this study, we used a custom oligonucleotide microarray to determine the overall
transcriptional response of laboratory strain (H37Rv) and most prevalent clinical strains (S7 and S10) of
M. tuberculosis from South India to hypoxia.
The climbing vine kudzu, a member of the leguminous
pea family (Fabaceae), was introduced into the USA
from its native Asia in the 1800s. It was initially lauded
for efficacy in erosion control along highways and as a
high-quality grazing crop for livestock. P. montana var.
lobata has since become a truculent invasive, spreading
via vegetative runners and seed dispersal. Seven
million acres of the American southeast are now
plagued by this vine.
1. Knockout Rats via Embryo Microinjection
of Zinc-Finger Nucleases
Aron M. Geurts,1,2
* Gregory J. Cost,3
* Yevgeniy Freyvert,3
Bryan Zeitler,3
Jeffrey C. Miller,3
Vivian M. Choi,3
Shirin S. Jenkins,3
Adam Wood,4
Xiaoxia Cui,4
Xiangdong Meng,3
Anna Vincent,3
Stephen Lam,3
Mieczyslaw Michalkiewicz,1,2
Rebecca Schilling,1,2
Jamie Foeckler,3
Shawn Kalloway,3
Hartmut Weiler,1,2
Séverine Ménoret,5
Ignacio Anegon,5
Gregory D. Davis,4
Lei Zhang,3
Edward J. Rebar,3
Philip D. Gregory,3
Fyodor D. Urnov,3
Howard J. Jacob,1,2,6
† Roland Buelow7
†
T
he laboratory rat is a well-established
model for the genetic dissection of human
disease-related traits (1) despite the fact
that targeted modification of its genome is largely
intractable. We investigated the application of
engineered zinc-finger nucleases [ZFNs (2)] for
the elimination of specific rat gene functions and
generation of knockout rats. ZFNs induce site-
specific, double-strand DNA breaks that can be
repaired by the error-prone nonhomologous end-
joining DNA repair pathway to result in a targeted
mutation (Fig. 1A). In the fruit fly and zebrafish,
direct embryo injection of ZFN-encoding mRNA
has been used to generate heritable knockout mu-
tations at specific loci (2).
The design and validation of three sets of
ZFN reagents that target the green fluorescent pro-
tein (GFP) gene and two endogenous rat genes,
Immunoglobulin M (IgM) and Rab38, were per-
formed as described (3) and are detailed in (4). To
take advantage of the potential for greater speci-
ficity of action afforded by longer (and therefore
rarer) targets, we used five- and six-finger ZFNs.
We delivered these ZFNs to 36 hemizygous
GFP-transgenic (5) inbred SS (Dahl S; GFP ZFNs),
91 inbred FHH (Fawn-hooded hypertensive;
Rab38 ZFNs), and 2793 outbred SD (Sprague
Dawley; IgM ZFNs) embryos by pronuclear or
intracytoplasmic injection of ZFN-encodingDNA
or mRNA at different concentrations (table S1).
Screening 295 founder animals yielded 35 (12%)
that harbored targeted mutations.
Full knockout of the GFP transgene was
achieved because mutant animals lacked both
GFP expression and wild-type GFP sequence
(Fig. 1, B and C). Thirty-two IgM mutants and
the single Rab38 mutant carried 25 to 100% dis-
rupted target chromosomes (fig. S1). Sequence
analysis of 18 founders revealed deletion alleles
ranging from 3 to 187 base pairs; of note, one ani-
mal carried biallelic mutations in IgM (table S1).
Furthermore, ZFN-mediated gene disruption dem-
onstrated high fidelity for each target sequence
because no ZFN-induced mutations were detected
in target gene–disrupted animals at any of 20 pre-
dicted ZFN off-target sites (figs. S2 and S3). After
breeding to wild-type animals, one out of one GFP
and three out of four IgM mutations were trans-
mitted through the germline, one of which was
subsequently bred to homozygosity (table S1 and
fig. S4).
The high percentage of disrupted chromo-
somes demonstrates that ZFNs are active in
early rat embryos from three strains, leading to
both mono- and biallelic gene disruption. Al-
though we observed no cleavage at predicted off-
target sites, such events could be segregated away
from the desired mutation by backcrossing to the
parental strain. ZFN-driven gene disruption and
germline transmission can be accomplished in
4 months’ time, and ZFNs can be engineered
against a broad range of sequences (6, 7); this
strategy adds a valuable tool to an increasingly
powerful rat genetic toolbox, opening up a
range of new experiments and models of human
disease.
References and Notes
1. T. J. Aitman et al., Nat. Genet. 40, 516
(2008).
2. D. Carroll, Gene Ther. 15, 1463 (2008).
3. Y. Doyon et al., Nat. Biotechnol. 26, 702
(2008).
4. Materials and methods are available as
supporting material on Science Online.
5. M. Michalkiewicz et al., Am. J. Physiol. Heart Circ.
Physiol. 293, H881 (2007).
6. C. O. Pabo, E. Peisach, R. A. Grant, Annu. Rev.
Biochem. 70, 313 (2001).
7. A. Klug, Proc. Jpn. Acad. 81, 87 (2005).
8. We thank R. Jaenisch, R. Hammer,
P. Sullivan, and three anonymous referees
for helpful suggestions; D. Smoller and
E. Lanphier for support; E. Eastlund for the
Rab38 ZFN mRNA; R. DeKelver and R. Amora for
technical assistance; and Caliper Life Sciences,
Incorporated for excellent service. Supported
by NIH grants 5U01HL066579-08 and
5P01HL082798-03, a sponsored research
agreement between the Medical College of
Wisconsin and Sigma-Aldrich, and the
American Physiological Society Fellowship in
Physiological Genomics to A.M.G. The authors
are filing patents based on the results reported
in this paper.
Supporting Online Material
www.sciencemag.org/cgi/content/full/325/5939/433/DC1
Materials and Methods
Figs. S1 to S5
Tables S1 and S2
References
18 February 2009; accepted 1 May 2009
10.1126/science.1172447
BREVIA
1
Human and Molecular Genetics Center, Medical College of
Wisconsin, Milwaukee, WI 52336, USA. 2
Department of Phys-
iology, Medical College of Wisconsin, Milwaukee, WI 52336,
USA. 3
Sangamo BioSciences, Incorporated, Richmond, CA 94804,
USA. 4
Sigma-Aldrich Biotechnology, St. Louis, MO 63103, USA.
5
INSERM, UMR 643, CHU, Nantes, Université de Nantes, 44322
Nantes, France. 6
Department of Pediatrics, Medical College of
Wisconsin, Milwaukee, WI 52336, USA. 7
Open Monoclonal
Technology, Incorporated, Palo Alto, CA 94303, USA.
*These authors contributed equally to this work.
†To whom correspondence should be addressed. E-mail:
jacob@mcw.edu (H.J.J.); rbuelow@omtinc.net (R.B.)
NT
Fig. 1. ZFN-mediated gene disruption in rat embryos. (A) ZFNs containing five or six fingers were
designed to target coding sequences of interest (gray lines) for site-specific cleavage. (B) Two of five
pups born after microinjection of GFP-targeted ZFNs were devoid of GFP expression. (C) Polymerase
chain reaction using GFP-specific primers revealed truncated but no wild-type sequence in each of the
GFP negative pups compared with positive littermates. SS indicates Dahl S control DNA; NT indicates no
template. (D) Table of injection data revealing successful mutagenesis of the three gene targets after
multiple delivery methods and doses in three rat strains.
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