312 CONSTRUCTION OF SPECIFICALLY EXPRESSED VECTOR IN MAMMARY GLAND FOR lacS AND ITS TRANSFECTION INTO BOVINE FETAL FIBROBLASTS MEDIATED BY LIPOSOME

2009 ◽  
Vol 21 (1) ◽  
pp. 253
Author(s):  
Z. H. Zhou ◽  
Z. J. Yan ◽  
L. J. Zhang
Keyword(s):  
Mammary Gland ◽  
Fluorescent Protein ◽  
Sulfolobus Solfataricus ◽  
Somatic Cells ◽  
Pcr Amplification ◽  
Cmv Promoter ◽  
Egfp Gene ◽  
Coding Sequence ◽  
Fetal Fibroblasts ◽  
Β Lactoglobulin

This study was designed to optimize conditions for transfection of a mammary gland specific transgene into bovine fetal fibroblasts. Transfection of Sulfolobus solfataricus β-glycosidase gene (lacS) was mediated by liposome. Neomycin resistance (Neor) and enhanced green fluorescent protein (EGFP) gene were used as genetic markers to screen transgenic somatic cells. A 0.92-kb fragment of bovine β-lactoglobulin gene sequence was obtained from bovine genome by PCR amplification and was inserted into the T site of pMD19-Simple T plasmid. 1.49 kb of lacS gene coding sequence was cloned from Sulfolobus solfataricus genome by PCR amplification and inserted into the pUC19 plasmid. The coding sequence of Neor was derived by PCR amplification from pIRES2-EGFP plasmid and inserted into the BamHI/NheI site of pIRES2-EGFP plasmid. The resultant vector (pNIE) contained a Neor and an EGFP gene, which were linked by an internal ribosome entry site sequence downstream of the cytomegalovirus (CMV) promoter. Finally, the vector pNIE was assembled into the pUC19 plasmid, thus creating a pBLI vector, which contained the Neor and EGFP gene regulated by CMV promoter for expression in a non-tissue specific mode and the lacS gene regulated by bovine β-lactoglobulin promoter for specific expression in mammary gland. Bovine fetal fibroblasts (bFF) were isolated from the ear skin of female fetuses at the age of 2 to 3 months. The cells proliferated well and grew normally in culture, with typical fibroblast morphology and growth curve. The effects of different concentrations of transfection and pBLI were compared on the efficiency of transfection. The passage 4 bFF cells at 70 to 80% confluency were transfected in a 24-well culture plate. 2 × 105 cells were cultured in DMEM with 0.5, 0.75, 1.0, 1.5, 2.0, and 2.5 μg of pBLI using transfection (1, 2, 3, 4, 5, and 6 μL) for 48 h, respectively. The transfected cells were cultured for 48 h before adding G418 at concentrations of 200, 300, 400, 500, 600, 700, 800, and 900 μg mL–1 for 14 d, respectively. Positive cell colonies were selected and purified through both the expression of Neor and EGFP gene under a fluorescence microscopy. The selected colonies were propagated in DMEM containing 300 μg mL–1 G418. The results showed that bright green fluorescence could be detected at 48 h after transfection. 1.0 μg of pBLI plasmid and 3 μL of transfection yielded the desirable efficiency of transfection. More transgenic bFF colonies were selected by G418 at the concentration of 800 μg mL–1. In conclusion, a specifically expressed vector in mammary gland for lacS gene was successfully constructed, transfection parameters were developed, and efficient screening measures were established for detecting transgenic somatic cells.

304 HUMAN AUGMENTER OF LIVER REGENERATION TRANSGENIC OVINE EMBRYO DERIVED FROM SOMATIC CELL NUCLEAR TRANSFER

2008 ◽  
Vol 20 (1) ◽  
pp. 232
Author(s):  
Y. Ma ◽  
P. Zhou ◽  
D. Liu ◽  
G. Xia ◽  
S. Bou
Keyword(s):  
Liver Regeneration ◽  
Nuclear Transfer ◽  
Genomic Sequence ◽  
Pcr Amplification ◽  
Foreign Gene ◽  
Green Fluorescence ◽  
Entry Site ◽  
Cmv Promoter ◽  
Coding Sequence ◽  
Augmenter Of Liver Regeneration

The aim of this research was to develop an efficient screening technique to detect transgenic ovine embryos using neomycin resistance (NeoR) and enhanced green fluorescence protein (EGFP) genes as genetic markers. A 0.8-kb fragment of the ovine β-lactoglobulin promoter sequence (BLG) and 1.8 kb of the human augmenter of liver regeneration (ALR) genomic sequence were derived by PCR amplification. These 2 fragments were inserted into the MCS of pGEM-7zf(+) plasmid; this vector was named p7Z-BA. The coding sequence of NeoR was derived by PCR amplification from the plasmid pIRES2-EGFP and was assembled into the MCS of the pIRES2-EGFP plasmid. The resultant vector (pNIE) contained a NeoR gene coding sequence and an EGFP coding sequence linked by an internal ribosome entry site (IRES) sequence downstream of the CMV promoter. The vector pNIE was excised as an NsiI-SspI fragment and inserted into the vector p7Z-BA. In the end, we had a vector named pNEA, which contained the NeoR gene and the EGFP gene regulated by a CMV promoter for expression in a non-tissue specific mode, and the human ALR gene regulated by the BLG promoter for expression specifically in mammary gland. Sheep fetal fibroblast (SFFB) cells were isolated by attachment of tissue pieces from the ear skin of a 1- to 2-month ovine fetus. Karyotypes of the cells at the third passage and after 15 passages were analyzed. The cells proliferated well and more than 72% of the cells maintained a diploid karyotype after 15 passages. Therefore, the SFFB cells are amenable for transgenic cloning manipulations. For transfection, third-passage SFFB cells at 70% confluency were transfected in a 100-mm dish with pNEA (0.5, 1.0, 2.0, 3.0, 5.0, and 7.0 µg) using Lipofectamine 2000 (2, 4, 6, 8, 10, and 12 µL; Invitrogen, Carlsbad, CA). Cells were checked 24 to 48 h after transfection under fluorescence microscopy for GFP expression, and G418 selection (800 µg mL–1) was applied at that time. After 2 weeks, selected colonies were counted and propagated in culture medium containing 300 µg mL–1 G418 for 2 to 3 passages and cryopreserved. A small portion of the cells was analyzed by PCR for gene integration. Bright green fluorescence could be detected 24 to 48 h after transfection. More colonies were selected when transfection parameters were 2 µg of DNA and 10 µL of Lipofectamine. The results of PCR detection showed that the foreign gene was integrated into the genome. A total of 612 oocytes were aspirated from 2- to 5-mm follicles of ovine ovaries collected from an abattoir; 78% of them were matured after 18 h in culture. Four hundred forty-three oocytes were enucleated, and 332 enucleated oocytes were treated for electrofusion with green fluorescence cells. Of these, 180 (54.2%) couplets were fused. A total of 172 reconstructed embryos were stimulated and cultured in vitro, 31 (18%) of which developed to the blastocyst stage, and 19 blastocysts expressed GFP. In conclusion, we established an effective method to select transgenic embryos formed by nuclear transfer using transfected donor cells.

207 EFFICIENT GENERATION OF MYOSTATIN PROMOTER MUTATIONS IN BOVINE EMBRYOS USING THE CRISPR/Cas9 SYSTEM

2017 ◽  
Vol 29 (1) ◽  
pp. 212
Author(s):  
C. A. Pinzon ◽  
M. Snyder ◽  
J. Pryor ◽  
B. Thompson ◽  
M. Golding ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Muscle Mass ◽  
Transforming Growth Factor ◽  
Fluorescent Protein ◽  
Pcr Amplification ◽  
Negative Regulator ◽  
Bovine Embryos ◽  
Myostatin Gene ◽  
Fetal Fibroblasts ◽  
Green Fluorescent

The myostatin gene or growth differentiation factor 8 is a member of the transforming growth factor-β superfamily that acts as a negative regulator of muscle growth. Mutations inactivating this gene occur naturally in Piedmontese and Belgian Blue cattle breeds, resulting in a dramatic increase in muscle mass, albeit with unwanted consequences of increased dystocia and decreased fertility. Modulation of muscle mass increase without the unwanted effects would be of great value for improving livestock growth and economic value of livestock. The objective of our work was to use the CRISPR-Cas9 genetic engineering tool to generate deletions of different elements in the myostatin promoter in order to decrease the level of expression and obtain an attenuated phenotype without the detrimental consequences of an inactivating mutation. To achieve this objective 4 different small guide RNA (sgRNA) targeting the promoter near the mutation were designed with PAM positions from transcription starting site of −1577, −689, −555, and −116. These sgRNA were cloned individually into the Cas9 plasmids (px461, and px462; Addgene®). These plasmids allow for a dual puromycin resistance (px462) and green fluorescent protein (px461) selection. We first tested the functionality of these sgRNA in vitro by co-transfecting bovine fetal fibroblasts with a combination of both plasmids (Set 1 = sgRNA 1–4; Set 2 = sgRNA 2–3). Cells were exposed to puromycin (0.2 µg mL−1) for 72 h, then single and mixed colonies positive for green fluorescent protein expression were separated for propagation. The DNA was extracted for PCR amplification of the targeted region. Multiple deletions and a few insertion events were observed after PCR, bands were cloned into TOPO® vector (Thermo Fisher Scientific, Waltham, MA, USA) and sequenced. Sequencing results confirmed the PCR products as insertions or deletions in the myostatin promoter region. We proceeded to modify the myostatin promoter directly in bovine zygotes. For this, IVF-derived zygotes were randomly assigned to 3 different treatment groups Set 1, Set 2, or Null (no sgRNA) for microinjections. Each zygote was injected with ~100 pL of trophectoderm buffer containing 50 ng µL−1 of total sgRNA, 10 ng µL−1 of Cas9 mRNA, and 30 ng µL−1 of Cas9 protein with 1 mg mL−1 of fluorescent dextran. Day 7 post-IVF blastocysts were lysed and DNA was extracted for PCR amplification of the target region. In Set 1, 16 of 19 embryos (94.12%) were successfully edited, whereas in Set 2 there were 11 of 17 embryos (64.7%) edited. In both sets of sgRNA there was a high degree of mosaicism, with only 1 embryo demonstrating a homozygous deletion. In conclusion, CRISPR/Cas9 acts over the course of the first few cleavage divisions Further research is necessary to refine the CRISPR/Cas9 system for inducing genetic mutations in bovine embryos.

56 TRANSGENESIS BY HANDMADE CLONING USING EGFP-TRANSFECTED YUCATAN FIBROBLASTS

2007 ◽  
Vol 19 (1) ◽  
pp. 146
Author(s):  
P. M. Kragh ◽  
Y. Du ◽  
J. Li ◽  
Y. Zhang ◽  
L. Bolund ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Polar Body ◽  
Cumulus Cells ◽  
Miniature Pig ◽  
Egfp Gene ◽  
Fusion Procedure ◽  
Reconstructed Embryos ◽  
Fetal Fibroblasts ◽  
Handmade Cloning

Somatic cell nuclear transfer (SCNT) offers the possibility of pig transgenesis. Importantly, genetic manipulations can be performed in cells isolated from special breeds followed by SCNT into enucleated oocytes isolated from slaughterhouse ovaries. In the present study, we established production of Yucatan blastocysts by the handmade cloning (HMC) technique using non-transgenic fibroblasts from the Yucatan miniature pig, and produced transgenic blastocysts using enhanced green fluorescent protein (EGFP)-positive Yucatan fetal fibroblasts. For transgenesis, Yucatan fibroblasts from a 40-day old fetus were transfected with a vector containing an EGFP gene and a neomycin-resistance selection gene by lipofection. Well separated neomycin-resistant colonies were isolated, expanded, and used for HMC. For HMC, cumulus–oocyte complexes were aspirated from ovaries of slaughterhouse sows and matured for 41 h. Subsequently, the cumulus cells were removed in hyaluronidase, and zonae pellucidae were partially digested by incubation in pronase. Oocytes with a visible polar body (PB) were subjected to oriented bisection. Less than half of the cytoplasm adjacent to the PB was removed with a microblade. The remaining parts, i.e. cytoplasts, were used as recipients for embryo reconstruction. Reconstructed embryos were produced by a two-step fusion procedure. At the first step, one cytoplast was fused with one fibroblast in the absence of Ca2+. After one h, the cytoplast-fibroblast pair and another cytoplast were fused and activated simultaneously in the presence of Ca2+, and subsequently cultured in cytochalasin B and cycloheximide for 4 h. The development of reconstructed embryos to the blastocyst stage was determined after 7 days of in vitro culture. When using non-transgenic and EGFP-positive Yucatan fetal fibroblasts, the rate of blastocyst formation (mean � SEM) were 36 � 7% (36/102) and 42 � 7% (32/77), respectively. In conclusion, the HMC technique was very efficient for production of blastocysts of Yucatan miniature pig origin using both non-transgenic and EGFP-positive fetal fibroblasts.

409 EFFICIENCY OF CO-TRANSFECTION OF TRANSGENE AND Neor GENE INTO GOAT AND PIG FETAL FIBROBLASTS

2007 ◽  
Vol 19 (1) ◽  
pp. 320
Author(s):  
Y. M. Shin ◽  
S. M. Chang ◽  
B. C. Kim ◽  
C. S. Park ◽  
D. I. Jin
Keyword(s):  
Transfection Efficiency ◽  
Somatic Cells ◽  
Pcr Amplification ◽  
Pcr Analysis ◽  
Fibroblast Cells ◽  
Drug Resistant ◽  
Fetal Fibroblast ◽  
Resistant Genes ◽  
G418 Selection ◽  
Fetal Fibroblasts

Transgenic animals can be generated by nuclear transfer with genetically modified somatic cells in which the essential procedure of transgene transfection is required. Most transgene vectors are constructed to contain transgene and drug-resistant genes to enrich for somatic cells in which transgene integration has occurred. However, construction of transgene vectors along with drug-resistant genes may not be easy, due to inappropriate restriction sites. Therefore, in this study, two separate constructs, human tPA cDNA fused to β-casein promoter sequence as a transgene vector and neomycin-resistant gene (Neor) driven by PGK promoter as a drug-selectable gene, were co-transfected into pig and goat fetal fibroblast cells to estimate the efficiency of transgene transfection following G418 selection. First, goat fetal fibroblasts (GFF) and pig fetal fibroblasts (PFF) were tested for G418 resistance with different concentrations of G418. The pertinent concentrations of G418 were 800 µg mL−1 for GFF and 200 µg mL−1 for PFF. The linearized tPA vector and Neor gene vector were co-transfected into goat fetal fibroblasts and pig fetal fibroblasts with FuGENE6 transfection reagent (Roche Diagnostics, Mannheim, Germany). The cells were selected following exposure of 800 µg mL−1 and 200 µg mL−1 G418 for GFF and PFF, respectively, for 14 days. Cell colonies surviving G418 selection were assayed by PCR amplification with tPA-specific primers. Initially 2 × 106 GFF and PFF were transfected. Resistant colonies were counted and transferred to 24-well plates for expansion and PCR analysis. The results of co-transfection experiments are summarized in Table 1. The transfection of 2 × 106 GFF and PFF yielded an estimated 96 and 93 colonies, respectively, which survived as the G418 selection. However, 54 colonies of GFF and 39 colonies of PFF proliferated during expansion and were subjected to PCR analysis. Twenty-three and 5 of these colonies were identified to contain tPA transgene in GFF and PFF colonies, respectively. Transfection frequencies for tPA gene were 42.6% and 12.8% in GFF and PFF, respectively. These results suggest that co-transfection of transgene vector with Neor gene can be an alternative method for transfection of transgenes into fetal fibroblast cells. Table 1. Transfection efficiency of goat fetal fibroblasts (GFF) and pig fetal fibroblasts (PFF) following co-transfection of tPA gene and Neor gene

Production of transgenic canine embryos using interspecies somatic cell nuclear transfer

Zygote ◽  
2011 ◽  
Vol 20 (1) ◽  
pp. 67-72 ◽  
Author(s):  
So Gun Hong ◽  
Hyun Ju Oh ◽  
Jung Eun Park ◽  
Min Jung Kim ◽  
Geon A. Kim ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Fluorescent Protein ◽  
Somatic Cells ◽  
Embryonic Stem ◽  
Transfected Cells ◽  
Fetal Fibroblasts ◽  
Transgenic Cells

SummarySomatic cell nuclear transfer (SCNT) has emerged as an important tool for producing transgenic animals and deriving transgenic embryonic stem cells. The process of SCNT involves fusion of in vitro matured oocytes with somatic cells to make embryos that are transgenic when the nuclear donor somatic cells carry ‘foreign’ DNA and are clones when all the donor cells are genetically identical. However, in canines, it is difficult to obtain enough mature oocytes for successful SCNT due to the very low efficiency of in vitro oocyte maturation in this species that hinders canine transgenic cloning. One solution is to use oocytes from a different species or even a different genus, such as bovine oocytes, that can be matured easily in vitro. Accordingly, the aim of this study was: (1) to establish a canine fetal fibroblast line transfected with the green fluorescent protein (GFP) gene; and (2) to investigate in vitro embryonic development of canine cloned embryos derived from transgenic and non-transgenic cell lines using bovine in vitro matured oocytes. Canine fetal fibroblasts were transfected with constructs containing the GFP and puromycin resistance genes using FuGENE 6®. Viability levels of these cells were determined by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay. Interspecies SCNT (iSCNT) embryos from normal or transfected cells were produced and cultured in vitro. The MTT measurement of GFP-transfected fetal fibroblasts (mean OD = 0.25) was not significantly different from non-transfected fetal fibroblasts (mean OD = 0.35). There was no difference between transgenic iSCNT versus non-transgenic iSCNT embryos in terms of fusion rates (73.1% and 75.7%, respectively), cleavage rates (69.7% vs. 73.8%) and development to the 8–16-cell stage (40.1% vs. 42.7%). Embryos derived from the transfected cells completely expressed GFP at the 2-cell, 4-cell, and 8–16-cell stages without mosaicism. In summary, our results demonstrated that, following successful isolation of canine transgenic cells, iSCNT embryos developed to early pre-implantation stages in vitro, showing stable GFP expression. These canine–bovine iSCNT embryos can be used for further in vitro analysis of canine transgenic cells and will contribute to the production of various transgenic dogs for use as specific human disease models.

scholarly journals Designing of a DNA matrix for transgene integration into the bovine beta-lactoglobulin gene locus using CRISPR/Cas9 technology

2020 ◽  
Vol 10 (5) ◽  
pp. 206-210
Author(s):  
E.M. Koloskova ◽  
V.A. Ezerskiy ◽  
K.S. Ostrenko
Keyword(s):  
Homologous Recombination ◽  
Fluorescent Protein ◽  
Transgenic Animals ◽  
Biologically Active ◽  
Cmv Promoter ◽  
Specific Expression ◽  
Egfp Gene ◽  
Genomic Editing ◽  
Beta Lactoglobulin

Beta-lactoglobulin (BLG) is the main protein in milk serum in almost all mammals, with the exception of rodents and primates. Regulatory regions of the beta-lactoglobulin gene in ruminants (sheep, goats, and cattle) as part of genetic constructs provide tissue - specific expression of recombinant protein in the mammary gland and have been actively used in genetic engineering since the beginning of the era of creating transgenic animals. To work effectively with the CRISPR/Cas9 genomic editing method, it is necessary to know the exact DNA sequence of the target gene: this is necessary both for creating a DNA matrix for homologous recombination and for the targeted accuracy of guide RNAs. A polymorphic variant of the bovine BLG gene was identified, whose sperm was used to fertilize cow oocytes in vitro. The aim of this work was to create a plasmid containing 5’ - and 3’ - arms of homology (ha) to the bovine BLG gene. Based on ??TZ57R/T, the pTZhaBLG plasmid was obtained, which has a unique site for EagI restriction at the junction of the homology arms. A fragment containing a biologically active protein gene can be embedded in the resulting plasmid at this restriction site. We created the pBLGcmvEGFP plasmid containing the green fluorescent protein (EGFP) gene under the cytomegalovirus (cmv) promoter: protein expression can serve as a reliable indicator of successful integration of the transgene into the genome. The resulting plasmids in circular or linearized form are intended for site-specific integration by homologous recombination repair into the BLG gene using CRISPR/Cas9 components.

scholarly journals The CRISPR/Cas9 Minipig—A Transgenic Minipig to Produce Specific Mutations in Designated Tissues

Cancers ◽  
2021 ◽  
Vol 13 (12) ◽  
pp. 3024
Author(s):  
Martin Fogtmann Berthelsen ◽  
Maria Riedel ◽  
Huiqiang Cai ◽  
Søren H. Skaarup ◽  
Aage K. O. Alstrup ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Somatic Cells ◽  
Genetic Alterations ◽  
Lung Epithelial Cells ◽  
Target Cells ◽  
Multiple Gene ◽  
Conditional Expression ◽  
Gene Alterations

The generation of large transgenic animals is impeded by complex cloning, long maturation and gastrulation times. An introduction of multiple gene alterations increases the complexity. We have cloned a transgenic Cas9 minipig to introduce multiple mutations by CRISPR in somatic cells. Transgenic Cas9 pigs were generated by somatic cell nuclear transfer and were backcrossed to Göttingen Minipigs for two generations. Cas9 expression was controlled by FlpO-mediated recombination and was visualized by translation from red to yellow fluorescent protein. In vitro analyses in primary fibroblasts, keratinocytes and lung epithelial cells confirmed the genetic alterations executed by the viral delivery of single guide RNAs (sgRNA) to the target cells. Moreover, multiple gene alterations could be introduced simultaneously in a cell by viral delivery of sgRNAs. Cells with loss of TP53, PTEN and gain-of-function mutation in KRASG12D showed increased proliferation, confirming a transformation of the primary cells. An in vivo activation of Cas9 expression could be induced by viral delivery to the skin. Overall, we have generated a minipig with conditional expression of Cas9, where multiple gene alterations can be introduced to somatic cells by viral delivery of sgRNA. The development of a transgenic Cas9 minipig facilitates the creation of complex pre-clinical models for cancer research.

scholarly journals Comparative AAV-eGFP Transgene Expression Using Vector Serotypes 1–9, 7m8, and 8b in Human Pluripotent Stem Cells, RPEs, and Human and Rat Cortical Neurons

2019 ◽  
Vol 2019 ◽  
pp. 1-11 ◽  
Author(s):  
Thu T. Duong ◽  
James Lim ◽  
Vidyullatha Vasireddy ◽  
Tyler Papp ◽  
Hung Nguyen ◽  
...  
Keyword(s):  
Cortical Neurons ◽  
Transgene Expression ◽  
Fluorescent Protein ◽  
Ex Vivo ◽  
Cell Types ◽  
Egfp Expression ◽  
Cell Type ◽  
Egfp Gene ◽  
Rat Cortical Neurons

Recombinant adeno-associated virus (rAAV), produced from a nonpathogenic parvovirus, has become an increasing popular vector for gene therapy applications in human clinical trials. However, transduction and transgene expression of rAAVs can differ acrossin vitroand ex vivo cellular transduction strategies. This study compared 11 rAAV serotypes, carrying one reporter transgene cassette containing a cytomegalovirus immediate-early enhancer (eCMV) and chicken beta actin (CBA) promoter driving the expression of an enhanced green-fluorescent protein (eGFP) gene, which was transduced into four different cell types: human iPSC, iPSC-derived RPE, iPSC-derived cortical, and dissociated embryonic day 18 rat cortical neurons. Each cell type was exposed to three multiplicity of infections (MOI: 1E4, 1E5, and 1E6 vg/cell). After 24, 48, 72, and 96 h posttransduction, GFP-expressing cells were examined and compared across dosage, time, and cell type. Retinal pigmented epithelium showed highest AAV-eGFP expression and iPSC cortical the lowest. At an MOI of 1E6 vg/cell, all serotypes show measurable levels of AAV-eGFP expression; moreover, AAV7m8 and AAV6 perform best across MOI and cell type. We conclude that serotype tropism is not only capsid dependent but also cell type plays a significant role in transgene expression dynamics.

scholarly journals Determination of the Absolute Number of Transgene Copies in CMVFUT Transgenic Pigs

2012 ◽  
Vol 12 (3) ◽  
pp. 349-356 ◽  
Author(s):  
Daniel Lipiński ◽  
Joanna Zeyland ◽  
Andrzej Pławski ◽  
Ryszard Słomski
Keyword(s):  
Copy Number ◽  
Transgenic Animals ◽  
Promoter Sequence ◽  
Absolute Number ◽  
Transgenic Pigs ◽  
Cmv Promoter ◽  
Genetic Construct ◽  
Coding Sequence ◽  
The Absolute

Determination of the Absolute Number of Transgene Copies in CMVFUT Transgenic PigsThe aim of this research was to determine the number of transgene copies in the DNA of transgenic pigs. The copy number of the transgene was analysed in the transgenic animals with introduced pCMVFUT genetic construct containing a coding sequence of human H transferase under a control of CMV promoter. The copy number of the transgene that had integrated with the genome of the transgenic animals was analysed by qPCR with SYBR Green dye, which enabled nonspecific double-stranded DNA detection. CMVFT-2F and CMVFT-2R primers were used to amplify a 149 bp fragment of DNA. Forward primer had a sequence complementary to a promoter sequence and reverse primer to a coding sequence of H transferase. The copy number of the transgene in the examined samples was established by plotting the CT values obtained on a standard curve, which had been set by the usage of the CT values for the successive standard dilutions with known copy number (1.438-1.431 copies). As a standard we used pCMVFut genetic construct hydrolyzed with Not I restriction enzyme to a linear form. The real-time PCR results helped to establish the range of 3 - 4 as the number of the transgene copies that had integrated to the swine genome.

Riboflavin uptake transporter Slc52a2 (RFVT2) is upregulated in the mouse mammary gland during lactation

2016 ◽  
Vol 310 (7) ◽  
pp. R578-R585 ◽  
Author(s):  
Alex Man Lai Wu ◽  
Liana Dedina ◽  
Pooja Dalvi ◽  
Mingdong Yang ◽  
John Leon-Cheon ◽  
...  
Keyword(s):  
Mammary Gland ◽  
Breast Milk ◽  
Milk Fat ◽  
Fluorescent Protein ◽  
Mammary Epithelial Cells ◽  
Mdck Cells ◽  
Basolateral Membrane ◽  
Mouse Mammary Gland ◽  
Transport Systems ◽  
Human Breast Milk

While it is well recognized that riboflavin accumulates in breast milk as an essential vitamin for neonates, transport mechanisms for its milk excretion are not well characterized. The multidrug efflux transporter ABCG2 in the apical membrane of milk-producing mammary epithelial cells (MECs) is involved with riboflavin excretion. However, it is not clear whether MECs possess other riboflavin transport systems, which may facilitate its basolateral uptake into MECs. We report here that transcripts encoding the second ( SLC52A2) and third ( SLC52A3) member of the recently discovered family of SLC52A riboflavin uptake transporters are expressed in milk fat globules from human breast milk. Furthermore, Slc52a2 and Slc52a3 mRNA are upregulated in the mouse mammary gland during lactation. Importantly, the induction of Slc52a2, which was the major Slc52a riboflavin transporter in the lactating mammary gland, was also observed at the protein level. Subcellular localization studies showed that green fluorescent protein-tagged mouse SLC52A2 mainly localized to the cell membrane, with no preferential distribution to the apical or basolateral membrane in polarized kidney MDCK cells. These results strongly implicate a potential role for SLC52A2 in riboflavin uptake by milk-producing MECs, a critical step in the transfer of riboflavin into breast milk.

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