1,721,003 research outputs found
Updating the zona-free method for mouse cloning using hm1 embryonic stem cells
No full textThe zona-free method of SCNT designed for bovine and pig cloning (Booth et al. 2001; Vajta et al. 2001; Oback et al. 2003) was successfully used for horse (Galli et al. 2003). Although simple and efficient in farm animals, its application in the mouse met several problems (Ribas et al. 2005, 2006). The aim of our work was to produce cloned mice using HM1 embryonic stem (ES)cells adapting a zona-free method. Seven- to 24-week-old superovulated B6D2F1 female mice were used as oocytes donors. Cumulus cells were removed by 0.3% hyaluronidase and the zona pellucida by 0.5% pronase in KSOM-HEPES (KSOM-H) 1 h later (Ribas et al. 2006) or immediately after hyaluronidase treatment at 37°C. The HM1 ES cells were cultured in KnockOut DMEM supplemented with leukemia inhibitory factor and 15% fetal bovine serum with or without 2i (Ying et al. 2008) and were synchronized at M phase by 3 ng mL–1 nocodazole for 3 h before fusion. Only spherical cells were selected for NT. Metaphase II chromosome spindle complexes were removed by micromanipulation in KSOM-H medium with 5 μg mL–1 cytochalasin B. Lectin-treated enucleated oocytes were attached to the donor cells in KSOM-H with nocodazole and fused by 2 pulses of 1.3 kV cm–1 DC for 30 μs in 0.3 M mannitol medium. Following 10- to 15-min incubation in KSOM-H, the fusion was assessed and repeated if the constructs were nonfused. Cloned embryos were activated in 1 mM SrCl2 in Ca2+-free KSOM medium for 2 to 2.5 or 5 to 6 h and cultured in 20-μL KSOM droplets using the well-of-the-well (WOW) method (Vajta et al. 2000) under mineral oil at 37°C and 5% CO2. Day 4 compacted morulae and blastocysts were surgically transferred into the uterus of Day-2.5 pseudopregnant recipients that were sacrificed on Day 19.5 to examine fetal development. The donor mice age was important for oocyte survival: ~16% of oocytes of 7- to 10-week-old mice lysed before or during fusion in 33% of experiments (n experiments = 15), whereas oocytes of older mice were not sensitive to enzymatic treatment and electric impulses even after 3 fusion rounds (n = 19). The time of pronase treatment did not affect oocyte survival, whereas extending the time between hyaluronidase treatment and enucleation revealed self-activation in ~25% of oocytes. The fusion efficiency of ES cells was significantly lower compared with serum-starved fibroblasts (61%, n = 623 v. 100%, n = 80). The duration of SrCl2 treatment did not affect embryo development (cleavage: 82% v. 84%; Day 4 blastocysts: 49% v. 52%). ES cell culture with 2i increased Day 4 blastocyst development (60.7% v. 50.4%; P = 0.07), and their ability to implant (52.6% v. 38.2%; P = 0.06). Moreover, only NT embryos derived from 2i-ES cells developed to term (8.2%, n = 5; P = 0.08), and produced live fetuses (4.9%, n = 3). In light of these results, the fusion of ES cells remains the critical step in the mouse zona-free protocol
Cell fusion in the liver, revisited
No full textThere is wide agreement that cell fusion is a physiological process in cells in mammalian bone, muscle and placenta. In other organs, such as the cerebellum, cell fusion is controversial. The liver contains a considerable number of polyploid cells: They are commonly believed to originate by genome endoreplication, although the contribution of cell fusion to polyploidization has not been excluded. Here, we address the topic of cell fusion in the liver from a historical point of view. We discuss experimental evidence clearly supporting the hypothesis that cell fusion occurs in the liver, specifically when bone marrow cells were injected into mice and shown to rescue genetic hepatic degenerative defects. Those experiments-carried out in the latter half of the last century-were initially interpreted to show "transdifferentiation", but are now believed to demonstrate fusion between donor macrophages and host hepatocytes, raising the possibility that physiologically polyploid cells, such as hepatocytes, could originate, at least partially, through homotypic cell fusion. In support of the homotypic cell fusion hypothesis, we present new data generated using a chimera-based model, a much simpler model than those previously used. Cell fusion as a road to polyploidization in the liver has not been extensively investigated, and its contribution to a variety of conditions, such as viral infections, carcinogenesis and aging, remains unclear
Somatic cell nuclear transfer and transgenesis in large animals: current and future insights
No full textSomatic cell nuclear transfer (SCNT) was first developed in livestock for the purpose of accelerating the widespread use of superior genotypes. Although many problems still exist now after fifteen years of research owing to the limited understanding of genome reprogramming, SCNT has provided a powerful tool to make copies of selected individuals in different species, to study genome pluripotency and differentiation, opening new avenues of research in regenerative medicine and representing the main route for making transgenic livestock. Besides well-established methods to deliver transgenes, recent development in enzymatic engineering to edit the genome provides more precise and reproducible tools to target-specific genomic loci especially for producing knockout animals. The interest in generating transgenic livestock lies in the agricultural and biomedical areas and it is, in most cases, at the stage of research and development, with few exceptions that are making the way into practical applications
High throughput production of multi-transgenic pig for xenotranplantation research
No full textDue to the increasing demand of organ for transplantation and
the shortage of human donors several areas of research,
including regenerative medicine and xenotransplantation, are
currently explored to provide different clinical solutions to a
varieties of pathological conditions. While regenerative medicine
will relay on stem cell auto-regeneration and/or cell
transplantation in partially compromised tissues and organs,
xenotransplantation would provide ready to use tissues or
organs for more severe pathologies or organ failures. Genetic
engineering of porcine genome lies at the heart of xenotransplantation
research since the pig is currently considered, on the
risk/benefit ratio, the most appropriate species. The stepsinvolved in the creation of candidate animals for xenotransplantation
research include the selection of a somatic cell line
(usually fibroblasts) that is engineered using current technologies,
either for the inactivation or for the insertion of
candidate genes, followed by somatic cell nuclear transfer
(SCNT) procedure to generate live animals. The founder
animals obtained can be suitable directly for xenotransplantation
and/or their cells can be further engineered.
In our laboratory we have been working primarily with a
male GAL-KO miniature pig cell line (provided by D. Sachs,
MGH, Boston USA) with the aim of overexpressing under an
ubiquitous promoter (pCGACGS) several transgenes controlling
complement mediated lysis and inflammation (hCD55,
hCD39), and coagulation (hEPCR, hTM). To speed up the
process we cotransfected by nucleofection two transgenes at
the same time, one of which carried the selectable marker
(hCD55HygromycinR ? hCD39; hEPCRPuromycinR ? hTM).
Forty-eight hours later the cells were subjected to drug selection
for 15 days to isolate resistant cell clones that were expanded
in duplicates for SCNT and phenotypic characterisation by
Western blot and immunocitochemistry to assess the presence
of the protein and the uniform expression of the transgenes in all
morphologically normal cells. At this point the clones expressing
single or the double transgenes were selected for nuclear transfer.
Zona-free enucleated oocytes were fused with fibroblasts,
activated and cultured in vitro up to the blastocyst stage for
6 days. Blastocysts were subsequently transferred to synchronised
recipient sows on day 5 after ovulation. Pregnant animals
were allowed to go to term and farrowing was induced with
prostaglandin, if it did not occur by day 118 of gestation, or by
caesarian section.
After nucleofection of 106 cells with CD55-CD39 we
obtained 60 colonies of which 12 did not express the
transgenes, 15 expressed one and 21 expressed both transgenes
while 12 were composed of negative and positive cells
(variegated). After nucleofection of 106 cells with EPCRhTM
we obtained 12 colonies of which 3 did not express the
transgenes, 7 expressed one and 2 expressed both transgenes
however possessed some negative for EPCR cells. ICC
analyses matched the data of WB and gave possibility to see
the quality of the cells and the degree of uniform expression
(absence of variegation) and possible contamination of colonies
by negative cells.
For each recipient sow we implanted pools of 86 ± 12
(range 59–109) embryos coming from 4 cell colonies with high
expression either of CD55 alone or both CD55-CD39 to
optimise pregnancy rates and embryo survival. We transferred
embryos to 27 sows and obtained 6 out of 10 pregnancies for
the single and 7 out of 16 for the double transgene, but one of
the single and three with double transgenic embryos were lost
by day 40. Forty-nine piglets were obtained of which 28 were
alive at birth and 20 were alive after 1 week. The development
to term of the single transgenic CD55-embryos was higher in
comparison with double transgenic embryos (6 piglets/sow vs.
3.25 piglet/sow). The higher abortion rate and small number of
piglets per litter suggest the detrimental effect of ubiquitous
high CD39 expression in the donor cells. The WB analysis of
the umbilical cord of all animals and of some organs of the
stillborn confirmed the expression in all animals obtained with
some minor differences in the levels of the proteins. After first
PCR screenings, Southern Blot (SB) analysis done on CD55+ piglets live at birth confirmed that starting from 8 selected
fibroblasts colonies (used in two different pools) we obtained 6
different founder animals of which 5 were CD55+ positive. The
same strategy was used also on CD55+
-CD39+ piglets live at
birth identifying 3 different founders starting from 4 double
transgenic colonies.
In conclusion, we implemented a system that, by combining
thorough analysis of the transfected donor fibroblast
cell clone for uniformity and high level of protein expression
and pooling embryos of different clonal origin, can efficiently
and reliably generate founder animals in sufficient number to
be used directly for xenotransplantation. Moreover particularly
interesting phenotypes can be re-cloned for breeding
purpose
Naturally-occurring porphyrins in a spontaneous-tumour bearing mouse model
No full textAn increase in naturally-occurring porphyrins has been described in the blood of subjects bearing different kinds of tumours, that has been proposed as an additional parameter for the diagnosis of occult cancer, although at present the reason for the phenomenon is not exactly defined. In this work the increase of porphyrins in plasma of tumour-bearing subjects has been investigated in parallel with their occurrence in other tissues, considering the systemic iron homeostasis subversion taking place in the presence of cancer. The transgenic female MMTV-neu mouse-developing spontaneous mammary adenocarcinoma has been used as an experimental model, in comparison to non-transgenic C1 mouse as a control. The spleen, accomplishing both hemocatheretic and hemopoietic functions in rodents, and the liver have been considered because of their deep engagement in heme metabolism, entailing both the fate of protoporphyrin IX (PpIX) as its ultimate precursor, and iron homeostasis. Investigations have been performed by means of microspectrofluorometric and image analysis of tissue autofluorescence (AF), and histochemical detection of non-heme iron. In tumour-bearing mouse, along with a marked PpIX presence in tumour, a PpIX enhancement in spleen and liver is observed, that is accompanied by a significant increase in plasma. The phenomenon can be related to a systemic alteration of heme metabolism induced by tumour cells to face their survival and proliferation requirements
EFFICIENT EXPRESSION OF HUMAN ENDOTHELIAL PROTEIN C RECEPTOR AND HUMAN THROMBOMODULIN IN TRANSFECTED PIG PRIMARY hCD55(+)-GAL(-/-) FIBROBLASTS USING F2A EXPRESSION VECTOR
No full textThe genetic engineering of the pig genome for xenotransplantation studies requires the insertion of different transgenes to create multi-transgenic pigs. In order to simultaneously add more transgene in a single genetic insertion, we constructed a polycistronic vector using the F2A self-cleaving peptide. Moreover, this solution has the added advantages of preventing possible segregation during breeding of the animals and of guaranteeing an equimolar production of chosen transgenes. The scope of this work was the construction and validation of an ubiquitous F2A-bicistronic expression vector for human thrombomodulin (hTM) and human endothelial protein C receptor (hEPCR) genes in pig primary hCD55-GAL–/– cells to establish transgenic fibroblasts colonies, to be used for somatic cell nuclear transfer (SCNT) to generate pigs for xenotransplantation research. The expression vector consisted of pCAGGS promoter (CMV-IE+chicken β actin) followed by hEPCR-furinF2A-hTM coding sequence. The resulting expression cassette was inserted between 2 insulators obtained from the 5′ MAR region of chicken lysozyme. Outside of this insulated structure, there is a loxable puromycin selection cassette. The resulting purified and linearized expression vector (pEFTM/Lgu I = 5 μg) was transfected into hCD55-GAL–/– primary fibroblasts (1 × 106), using Nucleofector (Amaxa, Lonza, Cologne, Germany), in parallel for comparative purposes we cotransfected the 2 pCAGGS-monocistronic vectors for the same transgenes (hEPCR and hTM = 1:3, 5 μg). Transfected cells were selected with puromycin (1 μg mL–1) for 15 days. After 8 days of selection, resistant colonies were picked up and expanded into 24-well plates for cryopreservation and analyses. Bicistronic transfection produced 20 clones and cotransfection only 8 clones that were analysed by Western blot (WB) and by immunocytochemistry (ICC) using polyclonal antibody anti-EPCR (1:250, R&D) and monoclonal antibody ab6980-Abcam (1:5000, Abcam, Cambridge, UK) in WB; polyclonal antibody RCR252 (1:100, Sigma-Aldrich, St. Louis, MO, USA) and monoclonal antibody ab6980-Abcam (1:100, Abcam) for ICC. Seventeen bicistronic clones (85%) and 2 cotransfected monocistronic clones (25%) were positive for both transgenes using WB. After ICC analyses, only 11 bicistronic colonies (55%) and 1 cotransfected colony (12.5%) uniformly expressed the desired transgenes and were selected for SCNT. The pCAGGS promoter maintained its strong expression also using the hEPCR-FurinF2A-hTM coding sequence and this bicistronic solution permitted us to improve our results obtained with co-transfection. Availability of hEPCR+ hTM+ hCD55+-GAL–/– colonies will allow us to obtain a new transgenic background for future xenotransplantation projects
Correction of a Recessive Genetic Defect by CRISPR-Cas9-Mediated Endogenous Repair
No full textCRISPR-Cas9 technology is a relatively recently developed tool for easy and efficient targeting of DNA. However, its efficiency for the repair of a mutated sequence is low. Moreover, most CRISPR-based gene correction approaches require the use of an exogenous template. Here, we investigated whether we could use the CRISPR-Cas9 system and the autologous repair machinery to correct human recessive genetic disorders having two different mutations in two alleles (compound heterozygotes). We reasoned that by targeting an intronic sequence located between the two mutations, we could generate at least one normal allele via the repair of induced double-strand breaks through either gene conversion or mitotic crossover. In particular, using a simple hypoxanthine-guanine phosphoribosyltransferase (Hprt)-based system, we show we can form a normal and functional Hprt gene. Thus, we give proof of principle that homology-directed recombination can be exploited in compound heterozygote cells to correct a genetic defect without exogenous templates
215 LIVE PIGLETS GENERATED BY SOMATIC CELL NUCLEAR TRANSFER FOLLOWING TARGETING OF A PORCINE ENHANCED GREEN FLUORESCENT PROTEIN LINE MEDIATED BY ZINC-FINGER NUCLEASES TO ESTABLISH CLONED HYGROMYCIN-RESISTANT PRIMARY CELL LINES SUITABLE FOR Cre-MEDIATED RECOMBINASE-MEDIATED CASSETTE EXCHANGE
No full textRecently, site-specific nucleases (zinc-finger nucleases, ZFN; TAL effector nucleases; and CRISPR) emerged as powerful tools for gene modification of different cells types and enhanced green fluorescent protein (EGFP)-specific ZFN were successfully used in the rat (Geurtz et al. 2010) and in the pig (Watanabe et al. 2010; Whyte et al. 2010). Previously (Brunetti et al. 2008 Clon. Stem Cells), we generated an EGFP transgenic porcine line (Verro2GFP) characterised by a single integration of pCAGGS-EGFP cassette, high ubiquitous EGFP expression, Mendelian transgene transmission, and expression in F1. The aim of this work was to modify a transcriptionally active GFP-locus into one suitable for Cre-mediated recombinase-mediated cassette exchange (RMCE), using EGFP-specific ZFN. Homology arms for promoter-less targeting vector were derived from pCAGGS-EGFP vector (promoter fragment = left-homology-arm = LHA; polyA sequence = right-homology-arm = RHA). Cloning floxed (lox2272/lox5171) hygromycin resistance coding sequence between LHA and RHA sequences, we generated the targeting/RMCE vector (pB5′3′Hygro-PL) and its positive control (C+) for PCR set-up (100–1000 plasmid copies). Verro2GFP fibroblasts cultured in DMEM+M199(1 : 1) + 10% FCS, bFGF in 5% CO2, 5% O2, were transfected using Nucleofector (V-024 program). In ZFN-mediated gene targeting, 2 μg of each ZFN coding vector (Sigma-CompoZr®) and 2 μg of pB5′3′Hygro-PL/KpnI vector were used to “nucleofect” 1.4 × 106 Verro2GFP fibroblasts in 2 experiments. Transfected cells were plated in 20 Petri dishes (Ø = 150 mm) and cultured under hygromycin selection (200 μg mL–1) for 15 days. After 12 days of drug selection, 82 resistant colonies were picked up and expanded in 24 multiwell plates for SCNT. All colonies were PCR screened and 45 (54.9%) colonies were positive. Four colonies were used in zona-free SCNT experiments with 140 Day 6 compacted morulae/blastocysts transferred into 2 synchronized sows that both became pregnant. One pregnancy went to term and delivered 5 live animals and 5 stillborn with correct hygromycin cassette integration, detected by PCR. The PCR products were sequenced in 7 animals to verify integration of promoterless targeting vector and in all 7 sequenced samples we obtained a correct insertion without any substitution/deletion. Using hygromycin selection in these experiments, we demonstrated that ZFN-mediated gene targeting can be easily done with high efficiency and is compatible with living animals. Moreover, we have validated a feasible SCNT-tested platform for further Cre-mediated site-specific gene modifications
Successful double genetic modification of porcine EGFP primary cell line using ZFN and Cre-mediated cassette exchange
No full textWe previously generated EGFP transgenic porcine line (Verro2GFP)
characterized by a single integration of pCAGGSEGFP
cassette, high ubiquitous EGFP expression, mendelian
transgene transmission and expression in F1 (Brunetti et al.
2008). Recently Zinc Finger Nucleases (ZFN) and TALENs
emerged as powerful tools for gene modification of different
cells types and Recombinase Mediated Cassette Exchange
(RMCE) was widely tested in different species. Moreover
EGFP-specific ZFNs were successfully used in rat (Geurtz
et al. 2010) and in pig (Watanabe et al. 2010, Whyte et al.
2010).
The purposes of our work were: a) to insert a vector suitable
for RMCE into a transcriptionally active locus and b) to assess
the possibility to obtain good quality cell colonies to be used in
Somatic Cell Nuclear Transfer (SCNT) even after two
sequential transgenic modifications (ZFN and RMCE), considering
the finite life span of Verro2GFP primary fibroblasts.
EGFP-specific ZFNs were purchased from Sigma (CompoZr®)
and the promoter-less targeting vectors were created using a
fragment of the promoter (Left-Homology-Arm = LHA) and the
polyA sequence (Right-Homology-Arm = RHA) of pCAGGSEGFP
expression vector. Cloning floxed (lox2272/lox5171)
reporter genes (PuromycinR, HygromycinR
) between these
homology sequences (LHA and RHA), we generated 2 targeting/exchanging
vectors (pB53Puro-PL, pB53Hygro-PL) and
their positive controls (C+). PCR on C+ was set up to obtain
minimal sensitivity of 100–1000 plasmid copies. Verro2GFP
fibroblasts (primary and colonies) cultured in DMEM + M199
(1:1) + FBS(10 %), 5 % CO2,5%O2, were transfected using
Nucleofector (V-24 program) and selected starting at 48 h after
transfection. Colonies were picked up at 8th selection day and
expanded in 24-well plates for PCR screening, RMCE and/or
SCNT. In ZFNs-mediated gene targeting, 2 μg of each ZFNs
coding vectors (pZFN1 and pZFN2) and 2 μg of pB53Puro-PL/
KpnI vector were used to “nucleofect” primary Verro2GFP
fibroblasts (3 105
; Puromycin = 1 μg/ml). In 3 experiments, 15
PuroR colonies were PCR screened, and 13 (87 %) were positive.
For subsequent RMCE, 4 colonies were pooled together and
4 105 cells were cotransfected with pB53Hygro-PL (2.5 μg)
and pCAG-CRE:EGFP (2.5 μg-Cre) vectors, and selected with
HygromycinB (175 μg/ml). One out of 3 HygroR colonies was positive. Finally 4 PuroR and 1 HygroR colonies were used in
zona-free SCNT to produce 243 reconstructed embryos, and 51
(21 %, 25 PuroR 26 HygroR
) blastocysts/compacted morulae
were transferred into two synchronized sows. These experiments
demonstrated that ZFN-mediated gene targeting following by
Cre-mediated cassette exchange can be successfully done during
the life span of Verro2GFP fibroblasts thus creating a feasible
platform for site-specific gene modification
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