Transgenic goats produced by DNA pronuclear microinjection of in vitro derived zygotes

2002 ◽  
Vol 63 (4) ◽  
pp. 437-443 ◽  
Author(s):  
Bin Wang ◽  
H. Baldassarre ◽  
T. Tao ◽  
M. Gauthier ◽  
N. Neveu ◽  
...  
Keyword(s):  
Pronuclear Microinjection ◽  
Transgenic Goats

State of the art in the production of transgenic goats

2004 ◽  
Vol 16 (4) ◽  
pp. 465 ◽  
Author(s):  
H. Baldassarre ◽  
B. Wang ◽  
C. L. Keefer ◽  
A. Lazaris ◽  
C. N. Karatzas
Keyword(s):  
Recombinant Proteins ◽  
Traditional Method ◽  
Nuclear Transfer ◽  
State Of The Art ◽  
Expression Vectors ◽  
Marketing Approval ◽  
Transfected Cells ◽  
Pronuclear Microinjection ◽  
Transgenic Goats

This review summarises recent advances in the field of transgenic goats for the purpose of producing recombinant proteins in their milk. Production of transgenic goats via pronuclear microinjection of DNA expression vectors has been the traditional method, but this results in low efficiencies. Somatic cell nuclear transfer has dramatically improved efficiencies in rates of transgenesis. Characterisation of transfected cells in vitro before use in nuclear transfer guarantees that kids born are transgenic and of predetermined gender. Using these platform technologies, several recombinant proteins of commercial interest have been produced, although none of them has yet gained marketing approval. Before these technologies are implemented in goat improvement programmes, efficiencies must be improved, costs reduced, and regulatory approval obtained for the marketing of food products derived from such animals.

Production of transgenic goats by pronuclear microinjection of in vitro produced zygotes derived from oocytes recovered by laparoscopy

2003 ◽  
Vol 59 (3-4) ◽  
pp. 831-839 ◽  
Author(s):  
Hernan Baldassarre ◽  
Bin Wang ◽  
Nacereddine Kafidi ◽  
Melanie Gauthier ◽  
Nathalie Neveu ◽  
...  
Keyword(s):  
Pronuclear Microinjection ◽  
Transgenic Goats

30 GENERATION OF CLONED TRANSGENIC GOATS WITH CARDIAC SPECIFIC OVEREXPRESSION OF TRANSFORMING GROWTH FACTOR β1

2013 ◽  
Vol 25 (1) ◽  
pp. 162 ◽  
Author(s):  
Q. Meng ◽  
J. Hall ◽  
H. Rutigliano ◽  
X. Zhou ◽  
B. R. Sessions ◽  
...  
Keyword(s):  
Growth Factor ◽  
Transforming Growth Factor ◽  
Critical Role ◽  
Transforming Growth Factor Β1 ◽  
Transgenic Goat ◽  
First Polar Body ◽  
Fetal Fibroblasts ◽  
Transgenic Goats ◽  
Tgf Β1

Transforming growth factor β1 (TGF-β1) has a potent profibrotic function and is central to signaling cascades involved in interstitial fibrosis, which plays a critical role in the pathobiology of cardiomyopathy and contributes to diastolic and systolic dysfunction. In addition, fibrotic remodeling is responsible for generation of re-entry circuits that promote arrhythmias (Bujak and Frangogiannis 2007 Cardiovasc. Res. 74, 184–195). Due to the small size of the heart, functional electrophysiology of transgenic mice is problematic. Large transgenic animal models have the potential to offer insights into conduction heterogeneity associated with fibrosis and the role of fibrosis in cardiovascular diseases. The goal of this study was to generate transgenic goats overexpressing an active form of TGFβ-1 under control of the cardiac-specific α-myosin heavy chain promoter (α-MHC). A pcDNA3.1DV5-MHC-TGF-β1cys33ser vector was constructed by subcloning the MHC-TGF-β1 fragment from the plasmid pUC-BM20-MHC-TGF-β1 (Nakajima et al. 2000 Circ. Res. 86, 571–579) into the pcDNA3.1D V5 vector. The Neon transfection system was used to electroporate primary goat fetal fibroblasts. After G418 selection and PCR screening, transgenic cells were used for SCNT. Oocytes were collected by slicing ovaries from an abattoir and matured in vitro in an incubator with 5% CO2 in air. Cumulus cells were removed at 21 to 23 h post-maturation. Oocytes were enucleated by aspirating the first polar body and nearby cytoplasm by micromanipulation in Hepes-buffered SOF medium with 10 µg of cytochalasin B mL–1. Transgenic somatic cells were individually inserted into the perivitelline space and fused with enucleated oocytes using double electrical pulses of 1.8 kV cm–1 (40 µs each). Reconstructed embryos were activated by ionomycin (5 min) and DMAP and cycloheximide (CHX) treatments. Cloned embryos were cultured in G1 medium for 12 to 60 h in vitro and then transferred into synchronized recipient females. Pregnancy was examined by ultrasonography on day 30 post-transfer. A total of 246 cloned embryos were transferred into 14 recipients that resulted in production of 7 kids. The pregnancy rate was higher in the group cultured for 12 h compared with those cultured 36 to 60 h [44.4% (n = 9) v. 20% (n = 5)]. The kidding rates per embryo transferred of these 2 groups were 3.8% (n = 156) and 1.1% (n = 90), respectively. The PCR results confirmed that all the clones were transgenic. Phenotype characterization [e.g. gene expression, electrocardiogram (ECG), and magnetic resonance imaging (MRI)] is underway. We demonstrated successful production of transgenic goat via SCNT. To our knowledge, this is the first transgenic goat model produced for cardiovascular research. This work was supported by the Utah Science Technology and Research Initiative, Utah Multidisciplinary Arrhythmia Consortium.

In vitro and in vivo developmental ability of the embryo derived from pronuclear microinjection using Piezo-micromanipulator in cattle

2009 ◽  
Vol 92 (3) ◽  
pp. S63
Author(s):  
F. Aono ◽  
K. Kawano ◽  
M. Kuwayama ◽  
Y. Takehara ◽  
K. Kato ◽  
...  
Keyword(s):  
Pronuclear Microinjection

In vitro assessment of the viability of sheep zygotes after pronuclear microinjection

1990 ◽  
Vol 2 (6) ◽  
pp. 633 ◽  
Author(s):  
SK Walker ◽  
TM Heard ◽  
PJ Verma ◽  
GE Rogers ◽  
CS Bawden ◽  
...  
Keyword(s):  
Salmonella Typhimurium ◽  
In Vitro Culture ◽  
Fluid Medium ◽  
Pronuclear Microinjection ◽  
In Vivo Culture ◽  
Developmental Capacity ◽  
In Vitro Assessment ◽  
Oviduct Fluid

Microinjected sheep zygotes were cultured in synthetic oviduct fluid medium (SOFM) for either 1 or 3 days and their subsequent developmental capacity was compared with that of microinjected zygotes cultured in vivo. Two experiments were carried out, using zygotes microinjected with one of three gene constructs containing the CysE and CysM genes from Salmonella typhimurium. In Experiment 1, microinjected zygotes were allocated to one of three treatments: (1) immediate transfer to recipient ewes (in vivo culture) followed by recollection 1 or 3 days later and subsequent transfer of viable embryos to other recipient ewes, (2) culture in SOFM (in vitro culture) for either 1 or 3 days before transfer to recipient ewes, and (3) immediate transfer to recipient ewes without subsequent interference. Recipient ewes were slaughtered on Day 14 of pregnancy and the number of elongated conceptuses determined. Although fewer zygotes failed to divide during in vitro culture than during in vivo culture, there were, overall, no significant differences between treatments in the percentage of zygotes that developed into elongated conceptuses (32.6-50.0%). In Experiment 2, microinjected zygotes were transferred immediately to recipient ewes or cultured in vitro for either 1 or 3 days before transfer. The number of fetuses per ewe on Day 50 of pregnancy and the number of lambs delivered per ewe were recorded. Neither the percentage of recipient ewes that became pregnant (overall 114/166, 68.7%) nor the percentage of zygotes that developed into lambs (overall 186/803, 23.2%) was significantly influenced by the culture treatment or by the gene construct microinjected.(ABSTRACT TRUNCATED AT 250 WORDS)

380 COMPARISON OF TRANSGENE EXPRESSIONS BY ICSI AND PRONUCLEAR MICROINJECTION IN MURINE AND PORCINE EMBRYOS

2006 ◽  
Vol 18 (2) ◽  
pp. 297
Author(s):  
H. Saito ◽  
H.-O. Kawano ◽  
M. Kurome ◽  
R. Tomii ◽  
S. Ueno ◽  
...  
Keyword(s):  
Transgene Expression ◽  
Expression Patterns ◽  
Gfp Expression ◽  
Pronuclear Microinjection ◽  
Significant Difference ◽  
Mosaic Embryo ◽  
Morula Stage ◽  
Transgenic Embryos

Intracytoplasmic sperm injection (ICSI) of DNA-binding sperm produces transgenic offspring as effectively as pronuclear microinjection (PNM). A significant difference in these two methods is that DNA is introduced into MII oocytes during ICSI, which is likely to allow earlier gene integration compared to PNM. This leads us to hypothesize that ICSI reduces the chance of development of a mosaic embryo, a mixture of transgene-positive and -negative cells. To test this hypothesis, we compared expression patterns of the green flourescent protein (GFP) gene introduced by ICSI and PNM into murine and porcine oocytes. For ICSI, 2 to 5 × 105/μL of sperm frozen-thawed in CZB (for mice) or NIM (for pigs) were co-incubated with 2.5 ng/μL of transgene fragments (CAG-EGFP; 3 kb) for 5 min. Murine sperm were microinjected into in vivo-matured oocytes, and porcine sperm into in vitro-matured oocytes. PNM was performed by microinjection of several picoliters of the transgene fragments (10 ng/μL) into pronuclei of in vivo-fertilized oocytes for mice and in vitro-matured and -fertilized oocytes for pigs. ICSI and PNM embryos were cultured in vitro to the morula stage and treated with 0.5% pronase to remove the zona pellucida. These morulae were disassembled into individual blastomeres by pipetting into PBS containing 100 μM EDTA and examined for GFP expression under fluorescence microscopy. As shown in Table 1, the rate of mosaicism in GFP-expressing embryos was significantly lower for ICSI than for PNM (P < 0.01). In addition, GFP-expressing ICSI embryos were likely to contain high percentages, 81 to 100%, of GFP-positive cells, whereas GFP-expressing PNM embryos were significantly less likely to contain such high percentages of GFP-positive cells (P < 0.01). From these results, we conclude that transgenesis by ICSI was less likely to produce mosaic embryos, and that produced transgenic embryos contained higher proportions of transgene-positive cells, although genomic integration remains to be determined. Table 1. Transgene expression by ICSI and pronuclear microinjection in murine and porcine embryos This work was supported by PROBRAIN.

408 FOREIGN GENE INTEGRATION PATTERNS IN TRANSGENIC PORCINE FETUSES PRODUCED BY ICSI-MEDIATED GENE TRANSFER

2007 ◽  
Vol 19 (1) ◽  
pp. 319
Author(s):  
H. Saito ◽  
M. Kurome ◽  
R. Tomii ◽  
S. Ueno ◽  
K. Hiruma ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Southern Blot ◽  
Disease Model ◽  
Foreign Gene ◽  
Fish Analysis ◽  
Pronuclear Microinjection ◽  
Model Study ◽  
Integration Sites ◽  
Random Site

We previously reported that transgenic (TG) pigs can be produced from in vitro-matured oocytes using intracytoplasmic sperm injection-mediated gene transfer (ICSI-mediated method) (Kurome et al. 2006 Transgenic Res. 15, 229–240). We subsequently studied the expression of a foreign gene which had been introduced by the ICSI-mediated method. We found that the ICSI-mediated method is considerably less likely than the pronuclear microinjection method to produce embryos in which transgene-positive and transgene-negative cells co-exist, that is, mosaic embryos (Saito et al. 2006 Reprod. Fertil. Dev. 18, 297 abst). Therefore, in order to further investigate the ICSI-mediated method, the present study was conducted to address the integration patterns of foreign genes introduced by this method. In particular, we wished to determine the number of transgene copies and number of chromosomal integration sites. TG pig fetuses, obtained by the ICSI-mediated method in a separate cardiac disease model study, were used in the present study. Porcine cumulus-oocyte complexes that had been collected from slaughterhouse ovaries were subjected to in vitro maturation in NCSU23 medium to produce MII oocytes to be used in this study. Porcine spermatozoa frozen in Beltsville Thawing Solution (BTS) were thawed rapidly in a 37�C water bath, and each spermatozoon was decapitated using ultrasound (28 kHz, 100 W; W-113; Honda Electronics Co., Ltd, Aichi, Japan). The heads (2 to 5 � 105/10 �L) were co-incubated with 2.5 ng �L-1 of rabbit calreticulin cDNA (�MHC-CRT-HA: 7.5 kb) for five min at room temperature, and then microinjected into MII oocytes using a piezo-micromanipulator. An electric stimulus (DC 150 V mm-1, 100 �s) was applied 10 to 40 min after microinjection in order to activate the oocytes. The embryos were cultured in PZM-5 medium for one to two days, and then transferred into the oviducts of recipient gilts, whose estrous cycle had been synchronized using 1000 IU eCG and 1500 IU hCG. Fetuses were collected 33 or 50 days later, and a primary cell line (fibroblast) was established. For each fetus, the number of transgene copies was determined by Southern blot. In addition, the chromosomal sites, where the foreign gene had integrated, were identified, and the number of integration sites was determined by fluoresent in situ hybridization (FISH). A total of 454 ICSI embryos were transferred to 4 recipients (92 to 135 embryos/recipient). All recipients became pregnant and 23 fetuses (5.1%, 23/454), including 7 TG fetuses (30.4%, 7/23), were obtained. Southern blot analysis showed that the number of transgene copies varied between 1 and 300 (1 copy: 1 fetus; 10 copies: 2 fetuses; 30 copies: 3 fetuses; 300 copies: 1 fetus). FISH analysis showed that in TG fetuses, the foreign gene had integrated at only a single chromosomal site, and this site varied from TG fetus to TG fetus. These results demonstrate that, in the case of ICSI-mediated gene transfer, as is the case for gene transfer by pronuclear microinjection, the integration patterns are: multiple copy, random site, and single site integration. This study was supported by PROBRAIN.

Selection of in vitro produced, transgenic embryos by nested PCR for efficient production of transgenic goats

2001 ◽  
Vol 56 (4) ◽  
pp. 545-556 ◽  
Author(s):  
S.Z Huang ◽  
Y Huang ◽  
M.J Chen ◽  
F.Y Zeng ◽  
Z.R Ren ◽  
...  
Keyword(s):  
Nested Pcr ◽  
Efficient Production ◽  
Transgenic Goats ◽  
Transgenic Embryos ◽  
Selection Of
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