Secretion of Biologically Active Human Granulocyte Colony-Stimulating Factor (G-CSF) in Milk of Transgenic Mice

Two constructs were devised, containing the full-length gene of the human granulocyte colony-stimulating factor (G-CSF) fused with the 5′ and 3′ flanking promoter sequences of bovine alpha-S₁-casein gene (CSN1S1). Both constructs contained a 1518-bp fragment that included exons 18 and 19 and 320 bp of the 3′ flanking region of bovine gene CSN1S1, but differed in size of the 5′ flanking sequences, which were of 721 bp, and exon 1 in construct pGCm1 and 2001 bp and exon 1 and intron 1 in construct pGCm2. With both constructs, transgenic mice were produced. The transgene expression was assessed using RT-PCR and immunochemically from the production of human G-CSF in milk of lactating females. Secretion of human G-CSF into the milk varied in a wide range, from 0.8 μg/ml to over 1 mg/ml, in mice with construct pGCm1 and was low (up to 60 μg/ml) or absent in mice with construct pGCm2. G-CSF glycosylation was incomplete in mice with transgene pGCm1 and complete in mice with pGCm2. G-CSF of transgenic mouse milk was shown to stimulate the formation and growth of granulocyte-containing colonies in human umbilical blood cell culture and be close or identical in physiological activity to the natural human G-CSF. __________ Translated from Genetika, Vol. 41, No. 10, 2005, pp. 1331–1337. Original Russian Text Copyright ? 2005 by Dvoryanchikov, Serova, Andreeva, Dias, Azevedo, Serov.     

In vitro transient expression system of latex C-serum was used for analysis of hevein promoter in response to abscisic acid in Hevea brasiliensis

Hevein has been found to be an essential element in coagulation of rubber particles in latex of rubber trees. In a previous study, we cloned a 1 241-bp fragment of a 5＇ upstream region of the hevein gene by genome walking. This fragment was analyzed by a 5＇ end nested deletion method in the present study, fused with a uidA （gus） gene to produce a series of tested constructs, which were transferred into C-serum of latex and the Gus activities were detected. Results showed that the fragment from -749 to -292 was sufficient for expression of gus gene in latex, and the fragment from -292 to -168 was crucial in response to abscisic acid inducement. In a transient transgenic test of rubber leaf with particle bombardment, construct Hev749 conferred gus-specific expression in veins, in which the latex tubes mainly distributed. This implies that the fragment from -749 to -292 was laticiferous-specific.     

Establishment of La-tPA/G-CSF dual transgenic mice and expression in their mammary gland

Expression vectors of human granulocyte colony stimulating factor (G-CSG) and long acting tissue plasminogen activator (La-tPA) in mammary gland were constructed using promoters of mouse whey acid protein gene (WAP) and sheep β-lactoglobulin gene (BLG) with sizes of 2.6 and 5 kb respectively. Two kinds of transgenic mice of G-CSF and La-tPA were produced with microinjection. The expression of G-CSF and La-tPA was achieved in mammary glands of transgenic mice, respectively. In order to establish dual transgenic mice of La-tPA/G-CSF, transgenic mice carrying G-CSF and La-tPA gene characterized with specific expression in mammary gland were mated. La-tPA/G-CSF dual transgenic mice were screened out from the hybrid offspring by Once-PCR. The co-expression of La-tPA and G-CSF in mammary gland of the dual transgenic mice was confirmed by the milk assayed and Northern blot analysis. Some parameters about the dual transgenic mice indicated that there were fewer litters than that of normal mice. The ratio of du     

An expression profile of human α-lactalbumin in the milk of transgenic mouse

Five female transgenic mice were produced by microinjection using a construct made up of a 7.3-kb-5′ flanking region and a 2.0-kb coding region of human α-lactalbumin, as well as a 227-bp 3′-flanking region from bovine growth hormone gene. A founder female expressed human α-lactalbumin as much as 0.3 g per liter of its milk, approximately a 3-fold increase in the total α-lactalbumin concentration of the transgenic mouse milk. Compared with the normal mice, the expression profile of the hα-Lac transgene in the transgenics is different during the lactation, showing low level in the first 3 days and becoming increased from day 4, then gradually reaching and stabilizing at the highest level from day 13. In addition, the milk yielding volume in the transgenics tended to be higher than in normal mice, suggesting higher concentrations of α-lactalbumin might boost more milk output.     

A protein in rat prostatic chromatin interacting with androgen regulated gene

2M NaCl-insoluble fraction of rat ventral Prostate chromatin(residual proteins)contain proteins able to interact specifically with androgen-receptor complex and is ,therefore,a part of the aceptor complex.Among residual proteins a 98 KDa protein has been found which binds significantly to a genomic fiagment containing an androgen-regulated gene coding for a 22 KDa protein The biological significance of this binding in androgen action need to be further studied.A mini-plasmid clone containing 22 KDa protein coding sequence was cloned into charon 4A genomic library from which a 5.7 Kb genomic fragment was isolated,identified by hybridization with a 5‘ and a 3‘ cDNA probes,and shown to contain the 3‘ flanking sequence.Restriction enzyme treatment of this fragment yielded a 4.7 Kb restriction fragmwent representing the 5‘ upstream region and a 1.0 Kb containing part of the coding sequence.Deletion studies indicated that the 97 KDa protein bound only to a subclone of about 300 bp segment .Furthermore,gel shifting experiment supported its DNA-protein binding.     

Comparative analysis of the pig BAC sequence involved in the regulation of myostatin gene

Myostatin (GDF-8, MSTN) is a member of trans- forming growth factors (TGF-β) superfamily, which was first described by McPherron et al. in 1997[1]. Myostatin appears to act as a negative regulator of muscle development and controls not only fibre size but also fibre number[2,3]. Mutations in the third exon of the myostatin gene have been shown to cause dou- ble muscling in cattle[4]. By knocking out the gene of myostatin in mice, they were able to show that the transgenic mice developed …     

Embryonic and genetic manipulation in fish

Fishes,the biggest and most diverse community in vertebrates are good experimental models for studies of cell and developmental biology by many favorable characteristics.Nuclear transplantation in fish has been thoroughly studied in China since 1960s.Fish nuclei of embryonic cells from different genera were transplanted into enucleated eggs generating nucleo-cytoplasmic hybrids of adults.Most importantly,nuclei of cultured goldfish kidney cells had been reprogrammed in enucleated eggs to support embryogenesis and ontogenesis of a fertile fish.This was the first case of cloned fish with somatic cells.Based on the technique of microinjection,recombinant MThGH gene has been transferred into fish eggs and the firsh batch of transgenic fish were produced in 1984.The behavior of foreign gene was characterized and the onsed of the foreign gene replication occurred between the blastula to gastrula stages and random integration mainly occurred at later stages of embryogenesis.This eventually led to the transgenic mosaicism.The MThGH-transferred common carp enhanced growth rate by 2-4 times in the founder juveniles and doubled the body weight in the adults.The transgenic common carp were more efficient in utilizing dietary protein than the controls.An “all-fish” gene construct CAgcGH has been made by splicing the common carp β-actin gene (CA) promoter onto the grass carp growth hormone gene (grGH) coding sequence.The CAgcGH-transferred Yellow River Carp have also shown significantly fast-growth trait.Combination of techniques of fish cell culture,gene transformation with cultured cells and nuclear transplantation should be able to generate homogeneous strain of valuable transgenic fish to fulfil human requirement in 21^st century.     

Can overexpression of TGF—β1 gene change the sex ratio in transgenic mice？

Mouse TGF-β1 gene was microinjected into male pronuclei of F2 hybrid fertilized eggs obtained by mating CSJLF1 and C57BL/6J inbred strains to generate transgenic mice with over-expressed TGF-β1 gene.The rate of founder production is 31% and Southern blot analysis of founder mice tail DNAs gave an integration efficiency of 33%.TGF-β1 gene could be stably integrated to the chromosomes of transgenic mice and transmitted to their progeny at a rate of 33% in the second generation.Dot blot analysis of tail RNA of some transgenic mice indicated a moderate expression of the transgene.The most interestin finding of the present work is the striking eviation from the normal male:female sex ratio in transgenic mice,with an average ratio of 6.7:1.The possible nature of the predominance of male sex in transgenic mice overexpressing TGF-β1 is discussed.     

羊BLG基因5′和3′调控区克隆及其调控GFP基因在乳腺细胞的表达

乳球蛋白 (ＢＬＧ)是反刍家畜的一种主要乳清蛋白质。将外源基因置于ＢＬＧ 5′调控区下游 ,能够控制外源基因在动物乳腺的组织特异性表达。现已清楚在ＢＬＧ 5′端有多个乳核因子 1(ＮＦ1)和转录因子Ｓｔａｔ5的识别位点 ,此外还存在多个激素作用位点[1 ] 。具有这些调控成分的ＢＬＧ 5′翼端 (5′ｆｌａｎｋｉｎｇ)能够控制外源基因在乳腺的表达 ,但受整合位点、外源基因结构以及转基因拷贝数及其排列方式的影响[2 ] 。研究结果表明 ,仅含有 5′调控区的乳腺表达构件 ,还不能使外源基因的表达达到内源性ＢＬＧ的表达水平 ,因为在 5′远端(…     

BLG-e1 - a novel regulatory element in the distal region of the beta-lactoglobulin gene promoter

beta-Lactoglobulin (BLG) is a major ruminant milk protein. A regulatory element, termed BLG-e1, was defined in the distal region of the ovine BLG gene promoter. This 299-bp element lacks the established cis-regulatory sequences that affect milk-protein gene expression. Nevertheless, it alters the binding of downstream BLG sequences to histone H4 and the sensitivity of the histone-DNA complexes to trichostatin A treatment. In mammary cells cultured under favorable lactogenic conditions, BLG-e1 acts as a potent, position-independent silencer of BLG/luciferase expression, and similarly affects the promoter activity of the mouse whey acidic protein gene. Intragenic sequences upstream of BLG exon 2 reverse the silencing effect of BLG-e1 in vitro and in transgenic mice.     

奶山羊β-乳球蛋白基因5＇、3＇调控区的克隆和序列分析

目的：克隆奶山羊β-乳球蛋白（BLG）基因5＇、3＇调控区,并对其进行序列分析。方法：从徐淮奶山羊组织中提取基因组DNA,采用高保真长模板PCR法克隆得到奶山羊BLG基因4.2kb的5＇调控区和1.8kb的3′调控区;将纯化后的PCR产物克隆到pGEM-TEasy载体中,经酶切鉴定和序列测定验证克隆的正确性。结果：部分序列分析表明,奶山羊BLG基因5＇区与GenBank中登录的山羊和绵羊BLG基因5＇区的同源性分别为100%和95%,3＇区的同源性分别为99%和93%;克隆得到的奶山羊BLG基因调控区与山羊BLG伪基因显著不同,二者5＇区和3＇区的同源性分别为83%和88%。结论：克隆得到的奶山羊BLG基因调控区可用于构建乳腺特异性表达载体,用于外源基因在转基因动物乳腺中的高效表达。     

Secretion of biologically active human granulocyte colony-stimulating factor (G-CSF) in milk of transgenic mice

Two constructs were devised, containing the full-length gene of the human granulocyte colony-stimulating factor (G-CSF) fused with the 5' and 3' flanking promoter sequences of bovine alpha-S1-casein gene. Both constructs contained a 1518-bp fragment that included exons 18 and 19 and 320 bp of the 3' flanking region of bovine gene @CSN1S1, but differed in size of the 5' flanking sequences, which were of 721 bp, and exon 1 in construct pGCm1 and 2001 bp and exon 1 and intron 1 in construct pGCm2. With both constructs, transgenic mice were produced. The transgene expression was assessed using RT-PCR and immunochemically from the production of human G-CSF in milk of lactating females. Secretion of human G-CSF into the milk varied in a wide range, from 0.8 microg/ml to over 1 mg/ml, in mice with construct pGCml and was low (up to 60 microg/ml) or absent in mice with construct pGCm2. G-CSF glycosylation was incomplete in mice with transgene pGCml and complete in mice with pGCm2. G-CSF of transgenic mouse milk was shown to stimulate the formation and growth of granulocyte-containing colonies in human umbilical blood cell culture and be close or identical in physiological activity to the natural human G-CSF.     

Accurate spatial and temporal transgene expression driven by a 3.8-kilobase promoter of the bovine β-casein gene in the lactating mouse mammary gland

The spatial, temporal, and hormonal pattern of expression of the β-casein gene is highly regulated and confined to the epithelial cells of the lactating mammary gland. Previous studies have shown that 1.7 kb of the bovine β-casein promoter were able to drive cell-specific and hormone-dependent expression to a mouse mammary cell line but failed to induce accurate expression to the mammary gland of transgenic mice. We investigated here the ability of 3.8 kb of the bovine β-casein gene promoter to drive the expression of the human growth hormone (hGH) gene in transgenic mice. A Northern blot analysis using total RNA obtained from different tissues of lactating and nonlactating females revealed the presence of hGH mRNA only in the mammary gland of lactating females. hGH mRNA was not detectable in the mammary gland of virgin females or males. A developmental analysis showed that hGH mRNA only peaked on parturition, resembling more closely the bovine β-casein temporal expression pattern rather than the murine. In situ hibridization studies performed on mammary gland sections showed that the cellular pattern of hGH expression was homogeneous in all lobules from heterozygous and homozygous transgenic mice. Silver grain counts on the tissue sections highly correlated with the hGH contents in the milk determined by radioimmunoassay (r = 0.996). Thus 3.8 kb of the bovine β-casein promoter direct a high-level expression of a reporter gene to the lactating mammary gland of transgenic mice in a tissue-specific and developmentally regulated manner. Mol. Reprod. Dev. 49:236–245, 1998. © 1998 Wiley-Liss, Inc.     

Elements within the beta-lactoglobulin gene inhibit expression of human serum albumin cDNA and minigenes in transfected cells but rescue their expression in the mammary gland of transgenic mice.

Two new beta-lactoglobulin (BLG)/human serum albumin (HSA) hybrid gene vectors were constructed and tested for expression in COS-7 cells and in transgenic mice. The HSA sequences were inserted between the second and sixth BLG exons. Transient transfection experiments with these vectors as well as a series of additional vectors with either the BLG 5'- or 3'- intragenic sequences revealed that sequences within BLG exon 1/intron 1/exon 2 abrogated BLG- directed HSA expression in vitro, regardless of the presence of HSA introns or the origin of the 3' polyadenylation signal. In contrast, the same BLG expression cassette enabled the efficient expression of HSA cDNA or minigene in the mammary gland of transgenic mice with subsequent secretion of the corresponding protein into the milk of 56 and 82%, respectively of the mouse strains at levels up to 0.3 mg/ml. Previous attempts to express HSA cDNA inserted into exon 1 of the BLG gene had failed [Shani,M., Barash,I., Nathan,M., Ricca,G., Searfoss,G.H., Dekel,I., Faerman,A., Givol,D. and Hurwitz,D.R. (1992) Transgenic Res. 1, 195- 208]. The new BLG expression cassette conferred more stringent tissue specific expression than previously described BLG/HSA constructs [Barash,I, Faerman,A., Ratovitsky,T, Puzis,R., Nathan,M., Hurwitz,D.R. and Shani, M. (1994) Transgenic Res. 3, 141-151]. However, it was not able to insulate the transgenes from the surrounding host DNA sequences and did not result in copy number dependent expression in transgenics. Together, the in vitro and in vivo results suggest both positive and negative regulatory elements within the BLG intragenic sequences evaluated. The new BLG construct represents an extremely valuable vector for the efficient expression of cDNAs in the mammary gland of transgenic animals.