Analysis of control elements for position-independent expression of human ?-lactalbumin YAC

Fujiwara, Yoshihiro; Takahashi, Riichi; Miwa, Masaichi; Kameda, Midori; Kodaira, Kunihiko; Hirabayashi, Masumi; Suzuki, Takashige; Ueda, Masatsugu

doi:10.1002/(sici)1098-2795(199909)54:1<17::aid-mrd3>3.0.co;2-n

Cited by 15 publications

(2 citation statements)

References 18 publications

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“…The use of YACs in transgenesis is likely to ensure position-independent, copy-number independent, and optimal levels of expression for transgenes, as long as all the regulatory elements are located within the vector (Giraldo and Montoliu, 2001). YACs have been used to produce transgenic mice, pigs (Yannoutsos et al, 1995), rabbits (Brem et al, 1996;Rouy et al, 1998), and rats (Fujiwara et al, 1997(Fujiwara et al, , 1999. In livestock, the benefits of YACs are mainly focused on xenotransplantation and the efficient production of recombinant proteins (Giraldo and Montoliu, 2001).…”

Section: Microchromosome Transfermentioning

confidence: 99%

437 Determining Gene Copy Number in Transfected Caprine Fibroblast Cells

Wilson¹,

Godke²,

Bondioli³

2010

Reprod. Fertil. Dev.

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Transgene expression in stably transgenic organisms is affected by many factors, including the copy number of the transgene in the genome and by interactions between the transgene and flanking DNA sequences. Very high transgene copy number has also been shown to affect genetic stability in transgenic plants and animals. Two commonly used methods for transfecting cells prior to their use in nuclear transfer (NT) are liposome-mediated transfection and electroporation. Little is known about the transgene copy number or variability of the copy number with these techniques. The objective of this study was to determine transgene copy number after liposome-mediated transfection and electroporation. The mean transgene copy number and variability between individual integration events have been determined. Q-PCR conditions were optimized for primer annealing temperature and concentration when amplifying a region of a plasmid expressing green fluorescent protein (GFP) under the control of the human elongation factor (hEF) promoter (hEFGFP) used for transfection. The quantitative nature of the Q-PCR reaction was confirmed by amplifying 10-fold dilutions of the plasmid and plotting the threshold cycle (CT) value against the log of the plasmid concentration. A correlation coefficient of 1.00 and a calculated PCR efficiency of 93.3% were obtained from this analysis. Caprine fibroblasts were transfected by electroporation with 20 μg of DNA or FuGENE® HD (Roche, Nutley, NJ, USA) reagent with 6 μg of DNA using either a circular or linearized hEFGFP plasmid. Transformed cells were plated at low density in medium containing Geneticin® (Gibco, Grand Island, NY USA). After 10 days of culture, single-cell colonies were isolated and expanded. When cultures reached 1 to 2 million cells, genomic DNA was isolated. Transgene copy number was determined by amplifying genomic DNA from individual clones representing 1 × 105 cells with Q-PCR. Transgene copy number was calculated from a standard curve of the transgene plasmid. The mean transgene copy number for electroporation circular was 2.7 ± 0.75 (n = 32 colonies) and 1.3 ± 0.65 (n = 19) when using a linear DNA construct. FuGENE HD using a circular plasmid construct generated a mean gene copy number of 0.5 ± 0.11 (n = 14) and 0.64 ± 0.13 (n = 16) for the linear plasmid construct. One-way ANOVA followed by multiple pair-wise comparisons using Tukey’s method showed significant differences when comparing electroporation circular to all other treatments. However, there were no differences when comparing electroporation linear, FuGENE HD circular, and FuGENE HD linear to each other. Because the calculated mean copy number for transfection with FuGENE HD was consistently less than 1, it is assumed that these colonies consisted predominantly of single-copy integrations. Our results indicate that the transfection method can affect gene copy number. Electroporation resulted in multiple but few copies whereas Fugene HD resulted in predominantly single-copy integrations. The probability of transgene mutation with single-copy integration suggests that electroporation is preferable forproducing transgenic animals by NT.

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Section: Microchromosome Transfermentioning

confidence: 99%

437 Determining Gene Copy Number in Transfected Caprine Fibroblast Cells

Wilson¹,

Godke²,

Bondioli³

2010

Reprod. Fertil. Dev.

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“…So far, Alac from various species were overexpressed only specifically in the lactating mammary gland of transgenic mammals using Alac-encoding gene regulatory sequences or, in one case, the αs1-casein promoter (Fujiwara et al, 1999). To assess if forced expression of this gene during early gestation or involution could affect mammary physiology, potentially highlighting the significance of some of the noncanonical functions attributed to Alac, a goat Alac-encoding minigene was expressed in transgenic mice under the transcriptional regulation of the mouse mammary tumor virus (MMTV) promoter derived from the pMSG expres- ; t1 = unspliced transgene-derived transcript; t2 = spliced transcript; t3 = unexpected shorter transcript; + = RT-PCR with addition of reverse transcriptase; -= PCR on product of reverse transcriptase reaction lacking reverse transcriptase enzyme, as negative control.…”

mentioning

confidence: 99%

Short communication: Mouse mammary tumor virus driven α-lactalbumin expression effects on lactation and fertility of transgenic mice

Guillou

Tilly

Passet

et al. 2011

Journal of Dairy Science

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α-Lactalbumin (Alac) is one of the major milk proteins. Its gene expression is restricted to epithelial cells of the lactating mammary gland. The Alac interaction with a uridine 5'-diphosphate-galactosyltransferase induces lactose synthesis, a major osmotic regulator of milk secretion. Other functions attributed to this protein include induction of apoptosis and anti-inflammatory activities. To assess if forced expression of this gene during early gestation or involution could affect mammary physiology, an Alac-encoding minigene was expressed in transgenic mice under the transcriptional regulation of the mouse mammary tumor virus promoter. The mammary expression did not interfere with gestation, resulted in a slight increase in milk yield as indirectly assessed by the 11% increased growth rate of the pups reared by transgenic females compared with that of those reared by control mice, and induced a slight delay in the early involution process, as demonstrated by histological analyses. The use of the mouse mammary tumor virus promoter resulted in Alac expression in several nonmammary tissues, such as the brain, the testis, the ovary, and the uterus. Although it did not affect male reproductive performances, it induced a female subfertile phenotype, characterized by embryonic implantation failure in the transgenic female reproductive tract.

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