Real-Time PCR to Determine Transgene Copy Number and to Quantitate the Biolocalization of Adoptively Transferred Cells from EGFP-Transgenic Mice

Joshi, Molishree; Pittman, H. Keith; Haisch, Carl E.; Verbanac, Kathryn M.

doi:10.2144/000112913

Cited by 68 publications

(59 citation statements)

References 26 publications

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“…The relative expression of EmGFP and b-casein was determined by qPCR. The primers used for detection of EmGFP expression were, forward 5'-CCACATGAAGCAGCACGACTT-3', reverse 5'-GGTGCGCTCCTGGACGTA-3' and probe 5'-FAM-TTCAAGTCCGCCATGCCCGAA-TAMRA-3', as reported by Joshi et al (2008), with replacement of one nucleotide in the forward primer. GAPDH expression was used as a control for endogenous gene expression.…”

Section: Lentiviral Production Bmec Transduction and Induction Of Emmentioning

confidence: 99%

β-casein gene expression by in vitro cultured bovine mammary epithelial cells derived from developing mammary glands

Monzani

Bressan²,

Mesquita³

et al. 2011

Genet. Mol. Res.

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ABSTRACT. Epithelial cells from mammary gland tissue that are cultured in vitro are able to maintain specific functions of this gland, such as cellular differentiation and milk protein synthesis. These characteristics make these cells a useful model to study mammary gland physiology, development and differentiation; they can also be used for production of exogenous proteins of pharmaceutical interest. Bovine mammary epithelial cells were cultured in vitro after isolation from mammary gland tissue of animals at different stages of development. The cells were plated on Petri dishes and isolated from fibroblasts using saline/EDTA treatment, followed by trypsinization. Cells isolated on plastic were capable of differentiating into alveolus-like structures; however, only cells derived from non-pregnant and non-lactating animals expressed β-casein. Real-time qPCR and epifluorescence microscopy analyses revealed that alveolus-like structures were competent at expressing Emerald green fluorescent protein (EmGFP) driven by the β-casein promoter, independent of β-casein expression.

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Section: Lentiviral Production Bmec Transduction and Induction Of Emmentioning

confidence: 99%

β-casein gene expression by in vitro cultured bovine mammary epithelial cells derived from developing mammary glands

Monzani

Bressan²,

Mesquita³

et al. 2011

Genet. Mol. Res.

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“…Quantitative Real-Time PCR Genomic DNA was extracted from brain slices 3 and 5 according to published methods (Joshi et al 2008), except that DNA was precipitated with 0.1x volume of 3 M sodium acetate and 2Â volumes of 100% ethanol. The purity and concentration of DNA was determined at 260 nm using a Nanodrop (ND-1000, version 3.7.0; Thermo Scientific, Scoresby, Australia).…”

Section: Sgsh Activity and Glcns-ua Measurement In Tissue Homogenatesmentioning

confidence: 99%

Neonatal Bone Marrow Transplantation in MPS IIIA Mice

et al. 2012

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Patients with some neurological lysosomal storage disorders (LSD) exhibit improved clinical signs following bone marrow transplantation (BMT). The failure of mucopolysaccharidosis (MPS) type IIIA patients and adult mice with the condition to respond to this treatment may relate to factors such as impaired migration of donorderived cells into the brain, insufficient enzyme production and/or secretion by the donor-derived microglial cells, or the age at which treatment is initiated. To explore these possibilities, we treated neonatal MPS IIIA mice with whole unfractionated bone marrow and observed that nucleated blood cell reconstitution occurred to a similar degree in MPS IIIA mice receiving green fluorescent protein (GFP)-expressing normal (treatment group) or MPS IIIA-GFP marrow (control group) and normal mice receiving normal-GFP marrow (control group). Further, similar distribution patterns of GFP + normal or MPS IIIA donor-derived cells were observed throughout the MPS IIIA mouse brain. We demonstrate that N-sulfoglucosamine sulfohydrolase (SGSH), the enzyme deficient in MPS IIIA, is produced and secreted in a manner proportional to that of other lysosomal enzymes. However, despite this, overall brain SGSH activity was unchanged in MPS IIIA mice treated with normal marrow and the lysosomal storage burden in whole brain homogenates did not decrease, most likely due to donor-derived cells comprising <0.24% of total recipient brain cells in all groups. This suggests that the failure of MPS IIIA patients and mice to respond to BMT may occur as a result of insufficient donor-derived enzyme production and/or uptake by host brain cells.

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“…This method of determining the transgene copy number may be used to overcome the limitations of Southern blot analysis. To date, quantitative PCR technology has been applied widely to analyze the copy number of transgenic animals, including mouse (Joshi et al, 2008;Vaisman, 2013) and pig (Watanabe et al, 2012;Ballester et al, 2013). However, thermal asymmetric interlaced PCR (TAIL-PCR), based on nest PCR and randomly primed PCR category (Liu and Chen, 2007), has become an extremely valuable and versatile tool for detecting insertion sites of foreign genes.…”

Section: Introductionmentioning

confidence: 99%

Copy number and integration sites in growth hormone transgenic goats

Zhang¹,

Lin²,

Yu³

et al. 2015

Genet. Mol. Res.

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ABSTRACT. Transgenic goats have been utilized for years to produce valuable protein. However, when transgenic goats are produced by random integration of inserted genes into cells, the copy number and integration sites of these genes in the goat genome are typically indefinite. Most polymerase chain reaction (PCR)-based methods that have been utilized to determine copy number and integration sites of inserted genes in the genome require complicated manipulations. In this study, we used quantitative real-time PCR and thermal asymmetric interlaced-PCR to determine copy number and integration sites of the inserted genes, respectively. Copies of transgenic goat lines GHcd-2 and GHcd-7 were 12.95 ± 0.18 and 12.24 ± 1.12, respectively. Two integration sites, located in chromosomes 3 and 11 and referred to as tg1 and tg2, were identified by thermal asymmetric interlaced-PCR. Junction PCR was then performed to confirm the integration sites of growth hormone transgenic goats. Transgenic copy number and integration sites were determined, which will be useful for determining the relationship between the growth hormone expression, copy number, and integration sites.

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Real-Time PCR to Determine Transgene Copy Number and to Quantitate the Biolocalization of Adoptively Transferred Cells from EGFP-Transgenic Mice

Cited by 68 publications

References 26 publications

β-casein gene expression by in vitro cultured bovine mammary epithelial cells derived from developing mammary glands

β-casein gene expression by in vitro cultured bovine mammary epithelial cells derived from developing mammary glands

Neonatal Bone Marrow Transplantation in MPS IIIA Mice

Copy number and integration sites in growth hormone transgenic goats

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