The molecular manipulation of milk composition

Genome ◽  
1989 ◽  
Vol 31 (2) ◽  
pp. 950-955 ◽  
Author(s):  
A. J. Clark ◽  
S. Ali ◽  
A. L. Archibald ◽  
H. Bessos ◽  
P. Brown ◽  
...  
Keyword(s):  
Mammary Gland ◽  
Dna Sequences ◽  
Germ Line ◽  
Fusion Gene ◽  
Milk Proteins ◽  
Factor Ix ◽  
Farm Animals ◽  
Specific Expression ◽  
Human Factor Ix ◽  
Β Lactoglobulin

The introduction of cloned genes into the mouse germ line is now routine. Although more difficult technically, gene transfer has been accomplished in farm animals and offers the potential for genetic improvement. In this regard, we have been investigating the use of transgenic animals as production vehicles for high value proteins in milk. We have shown that DNA sequences derived from the gene encoding sheep β-lactoglobulin mediate efficient and specific expression in the mammary gland. A fusion gene comprising β-lactoglobulin sequences and those encoding antihemophilic human factor IX has been constructed. This construct has been introduced into sheep; it is expressed in the mammary gland, and the corresponding protein is secreted into milk.Key words: transgenic, mice, sheep, milk, proteins.

339 BIOENGINEERING OF THE MAMMARY GLAND OF LIVESTOCK: INCREASED PROPEPTIDE PROCESSING OF FACTOR IX IN THE MILK OF TRANSGENIC PIGS BY CO-EXPRESSION OF FURIN

2011 ◽  
Vol 23 (1) ◽  
pp. 265 ◽  
Author(s):  
J. Zhao ◽  
E. Walters ◽  
J. Calcaterra ◽  
J. Ross ◽  
L. Spate ◽  
...  
Keyword(s):  
Mammary Gland ◽  
Production Efficiency ◽  
Coagulation Factor ◽  
Factor Ix ◽  
Coagulation Cascade ◽  
Blood Proteins ◽  
Wild Type ◽  
Transgenic Pigs ◽  
Human Factor Ix ◽  
Fetal Fibroblasts

Coagulation Factor IX is a vital protein that plays an important role in the blood coagulation cascade. Hereditary deficiency in the Factor IX gene can result in hemophilia type B, the second most common hemophilia. The milk of transgenic livestock can be an efficient vehicle for producing complex, post-translationally modified blood proteins. The advantages include decreased pathogen risk as well as 100-fold or more production efficiency over blood fractionation and cell culture biotechnology. Although the mammary gland can make many of the complex post-translational modifications necessary for biological function of blood proteins, improvements in propeptide cleavage and decreased proteolytic degradation are desirable. To explore the possibility of producing bioactive human Factor IX protein in pig milk, male and female Landrace fetal fibroblast cells were co-transfected by electroporation with 3 different transgene constructs, Factor IX (FIX), Furin, and SERPINA1. The SERPINA1 construct (containing a Neo selectable marker) was delivered into fetal fibroblasts at a 10-fold-lower molar concentration than the other 2 constructs. Following selection in Geneticin, the presence of all 3 genes was verified by PCR and then cells were used as donors for somatic cell nuclear transfer. Thirteen F0 female piglets from 3 potential different integrations were delivered, and 6 piglets were validated by PCR to be positive for all 3 genes. Among the 6 transgenic pigs, 3 are healthy and able to reach puberty. Milk was collected by induced lactation from 2 gilts. A short murine whey acidic protein promoter-Furin gene was used to limit Furin to the lowest levels needed for pro-FIX processing. Furin was expressed to increase propeptide cleavage efficiency, with the result being complete processing of pro-FIX to FIX at ∼0.3 g L–1 pro-FIX. Total FIX levels were ∼1 g L–1. SERPINA1 was also co-expressed at ∼1 g L–1 or more and this serine protease inhibitor did not seem to inhibit furin processing of the pro-FIX. Fifteen F0 male piglets from 3 potential different integration sites were delivered and all of them were positive for all 3 genes. Four F0 males were chosen to breed with wild-type females, and 5 litters of F1 piglets were born. Of 63 F1 piglets, 22 were tri-transgenic and 3 were di-transgenic (only carrying FIX and Furin). Two F1 females were mated with wild-type males, are confirmed to be pregnant, and will be used to determine the expression level and bioactivity of the Factor IX protein in the milk. Funded by the NIH NCRR (RR018877) and R01 HL078944.

scholarly journals Dynamic patterns of transcript abundance of transposable element families in maize

2019 ◽  
Author(s):  
Sarah N Anderson ◽  
Michelle C Stitzer ◽  
Peng Zhou ◽  
Jeffrey Ross-Ibarra ◽  
Cory D Hirsch ◽  
...  
Keyword(s):  
Transposable Elements ◽  
Dna Sequences ◽  
Germ Line ◽  
Transcript Abundance ◽  
Maize Genome ◽  
Rna Seq ◽  
Specific Expression ◽  
Poor Predictor ◽  
Repetitive Nature ◽  
Genomic Studies

AbstractTransposable Elements (TEs) are mobile elements that contribute the majority of DNA sequences in the maize genome. Due to their repetitive nature, genomic studies of TEs are complicated by the difficulty of properly attributing multi-mapped short reads to specific genomic loci. Here, we utilize a method to attribute RNA-seq reads to TE families rather than particular loci in order to characterize transcript abundance for TE families in the maize genome. We applied this method to assess per-family expression of transposable elements in >800 published RNA-seq libraries representing a range of maize development, genotypes, and hybrids. While a relatively small proportion of TE families are transcribed, expression is highly dynamic with most families exhibiting tissue-specific expression. A large number of TE families were specifically detected in pollen and endosperm, consistent with reproductive dynamics that maintain silencing of TEs in the germ line. We find that B73 transcript abundance is a poor predictor of TE expression in other genotypes and that transcript levels can differ even for shared TEs. Finally, by assessing recombinant inbred line and hybrid transcriptomes, complex patterns of TE transcript abundance across genotypes emerged. Taken together, this study reveals a dynamic contribution of TEs to maize transcriptomes.

The promoter region of the Drosophila alpha 2-tubulin gene directs testicular and neural specific expression

Development ◽  
1989 ◽  
Vol 106 (3) ◽  
pp. 581-587 ◽  
Author(s):  
J. Bo ◽  
P.C. Wensink
Keyword(s):  
Nervous System ◽  
Promoter Region ◽  
Germ Line ◽  
Fusion Gene ◽  
P Element ◽  
Tubulin Gene ◽  
Specific Expression ◽  
Spatial Expression Pattern ◽  
Males And Females ◽  
Temporal And Spatial

The temporal and spatial expression pattern of the Drosophila melanogaster alpha 2-tubulin gene (alpha 2) has been investigated by examining the expression of an alpha 2-lacZ fusion gene. When this fusion gene is introduced into the germ line by P-element mediated transformation, expression is only detected in chordotonal organs and testes. Chordotonal organs, which are sensory organs of the peripheral nervous system, express the gene from late embryonic through adult stages in both males and females. Testicular expression occurs from larval through adult stages and is limited to germ-line cells, the primary and secondary spermatocytes and perhaps the early spermatids.

199 Efficient Knock-out of Ovine β-Lactoglobulin (BLG) Gene and Knock-in of Recombinant Human Factor IX (rhFIX) Under BLG Native Regulatory Sequences in Somatic Cells and Zygotes Using TALEN Nuclease

2018 ◽  
Vol 30 (1) ◽  
pp. 240
Author(s):  
R. J. Bevacqua ◽  
D. Carlson ◽  
R. Fernandez-Martín ◽  
A. E. Gibbons ◽  
V. Savy ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Human Factor ◽  
Factor Ix ◽  
Regulatory Sequences ◽  
Knock Out ◽  
Human Factor Ix ◽  
Target Sites ◽  
Engineered Nucleases ◽  
Recombinant Human Factor Ix ◽  
Β Lactoglobulin

Site-specific genetic engineering is a valuable tool for pharmaceutical research and development of biomedical models. Despite engineered nucleases allow targeted gene edition in a rather simple fashion; few reports are available so far on specific gene knock-in (KI) combined with engineered nucleases in domestic species. Here, we evaluated the possibility of inducing specific KI of cDNAs coding for proteins of pharmaceutical interest under the control of milk native promoter sequences, taking advantage of the TALEN system, both in ovine somatic cells and in zygotes. We designed 2 TALENs, targeting exons 1 and 5 of ovine β-lactoglobulin gene (BLG), respectively, and a homologous recombination vector (pHR), carrying recombinant human factor IX (rhFIX) flanked by homology arms contiguous to the TALEN target sites. In an initial set of experiments, 5 × 105 to 1 × 106 ovine fibroblasts were transfected with 1 μg of each TALEN mRNA, with or without 50 ng μL−1 pHR. The feasibility of inducing knock-out (KO) was confirmed by Cel1 assay. The deletion of the genomic region between TALEN target sites and the occurrence of HR in cell lysates were assessed by PCR. Also, 576 individual colonies were picked up and analyzed by PCR. The deletion of the region between TALEN target sites was achieved with 7.8% efficiency (45/576). The incidence of HR in cells was 0.5% (3/576), as detected by PCR. In order to evaluate the system in zygotes, laparoscopic AI was performed on synchronized and superovulated ewes. Zygotes were recovered 16 h after AI and cytoplasmically injected with (1) 5 ng μL−1 TALEN mix (2.5 ng μL−1 oaBLG T1.1 + 2.5 ng μL−1 oaBLG T5.1) (5TM); (2) 5 ng μL−1 TALEN mix + 25 ng μL−1 pHR-hFIX plasmid (5TM+25pRH); or (3) 15 ng μL−1 TALEN mix (7.5 ng μL−1 oaBLG T1.1 + 7.5 ng μL−1 oaBLG T5.1) + 50 ng μL−1 pHR-hFIX (15TM+50pRH). A non-injected control (NIC) was also included. Embryo analysis was conducted on whole-genome amplified DNA from blastocysts, followed by PCR and sequencing. Non-parametric Fisher test was applied to detect significant differences among treatments. Although blastocyst rates for NIC and 5TM did not statistically differ, 5TM+25pRH and 15TM+50pRH groups resulted in lower blastocysts rates than the NIC [P < 0.05; 13/17 (76%), 6/15 (40%), 4/15 (26%) and 2/14 (14%) for NIC, 5TM, 5TM+25pRH and 15TM+50pRH respectively]. It was possible to detect the PCR product compatible with deletion of the entire region among TALEN target sites in 6/6 blastocysts (100%) from the group 5TM, 3/4 blastocysts (75%) from the group 5TM+25pRH and 2/2 (100%) blastocysts from the group 15TM+50pRH. HR was detected in 1/2 (50%) blastocysts injected with 15TM+50pRH and in 1/4 (25%) blastocysts injected with 5TM+25pRH, by PCR and sequencing of the PCR products. Our results indicate that TALEN combined with homologous recombination constitutes a powerful platform for the production of proteins of pharmaceutical interest under native regulatory sequences in the milk of genetically modified animals.

Differential regulation of rat beta-casein-chloramphenicol acetyltransferase fusion gene expression in transgenic mice

1989 ◽  
Vol 9 (2) ◽  
pp. 560-565
Author(s):  
K F Lee ◽  
S H Atiee ◽  
J M Rosen
Keyword(s):  
Gene Expression ◽  
Mammary Gland ◽  
Transgenic Mice ◽  
Fusion Gene ◽  
Chloramphenicol Acetyltransferase ◽  
Fusion Genes ◽  
Specific Expression ◽  
Casein Gene ◽  
Noncoding Exon ◽  
Beta Casein

Previous studies in our laboratory have demonstrated the mammary-specific expression of the entire rat beta-casein gene with 3.5 kilobases (kb) of 5' and 3.0 kb of 3' DNA in transgenic mice (Lee et al., Nucleic Acids Res. 16:1027-1041, 1988). In an attempt to localize sequences that dictate this specificity, lines of transgenic mice carrying two different rat beta-casein promoter-bacterial chloramphenicol acetyltransferase (cat) fusion genes have been established. Twenty and eight lines of transgenic mice carrying two fusion genes containing either 2.3 or 0.5 kb, respectively, of 5'-flanking DNA of the rat beta-casein gene along with noncoding exon I and 0.5 kb of intron A were identified, most of which transmitted the transgenes to their offspring in a Mendelian pattern. CAT activity was detected predominantly in the lactating mammary gland of female transgenic mice but not in the male mammary fat pad. A several-hundred-fold variation in the level of cat expression was observed in the mammary gland of different lines of mice, presumably due to the site of integration of the transgenes. CAT activity was increased in the mammary gland during development from virgin to midpregnancy and lactation. Unexpectedly, the casein-cat transgenes were also expressed in the thymus of different lines of both male and female mice, in some cases at levels equivalent to those observed in the mammary gland, and in contrast to the mammary gland, CAT activity was decreased during pregnancy and lactation in the thymus. Thus, 0.5 kb of 5'-flanking DNA of the rat beta-casein gene along with noncoding exon I and 0.5 kb of intron A are sufficient to target bacterial cat gene expression to the mammary gland of lactating mice.

Binding of a factor to an enhancer element responsible for the tissue-specific expression of the chicken alpha A-crystallin gene

Development ◽  
1991 ◽  
Vol 113 (2) ◽  
pp. 539-550 ◽  
Author(s):  
I. Matsuo ◽  
M. Kitamura ◽  
K. Okazaki ◽  
K. Yasuda
Keyword(s):  
Gene Expression ◽  
Dna Sequences ◽  
Nuclear Protein ◽  
Fusion Gene ◽  
Regulatory Region ◽  
Binding Activity ◽  
Flanking Sequence ◽  
Specific Expression ◽  
Enhancer Element

We have characterized a regulatory region of the chicken alpha A-crystallin gene using transfection assays, which revealed that a 84 base pair element (−162 to −79) in the 5′ flanking sequence is necessary and sufficient for lens-specific expression. A multimer of this element functions as lens-specific enhancer and synergistically activates transcription from chicken alpha A-crystallin or beta-actin basal promoters fused to the CAT gene. In vivo competition experiments demonstrated that DNA sequences containing the 84 bp element reduced alpha A-crystallin-CAT fusion gene expression. A nuclear factor present exclusively in lens cells binds to the 84 bp element in the region between positions −165 and −140. Southwestern blot analysis showed that 61,000 Mr (61 × 10(3) Mr) lens nuclear protein exhibited DNA-binding activity specific to the 84 bp element. Our data suggested that the 61 × 10(3) Mr nuclear protein, and the 84 bp element that it interacts with, may be involved in regulating the alpha A-crystallin gene expression in vivo.

scholarly journals Differential regulation of rat beta-casein-chloramphenicol acetyltransferase fusion gene expression in transgenic mice.

1989 ◽  
Vol 9 (2) ◽  
pp. 560-565 ◽  
Author(s):  
K F Lee ◽  
S H Atiee ◽  
J M Rosen
Keyword(s):  
Gene Expression ◽  
Mammary Gland ◽  
Transgenic Mice ◽  
Fusion Gene ◽  
Chloramphenicol Acetyltransferase ◽  
Fusion Genes ◽  
Specific Expression ◽  
Casein Gene ◽  
Noncoding Exon ◽  
Beta Casein

Previous studies in our laboratory have demonstrated the mammary-specific expression of the entire rat beta-casein gene with 3.5 kilobases (kb) of 5' and 3.0 kb of 3' DNA in transgenic mice (Lee et al., Nucleic Acids Res. 16:1027-1041, 1988). In an attempt to localize sequences that dictate this specificity, lines of transgenic mice carrying two different rat beta-casein promoter-bacterial chloramphenicol acetyltransferase (cat) fusion genes have been established. Twenty and eight lines of transgenic mice carrying two fusion genes containing either 2.3 or 0.5 kb, respectively, of 5'-flanking DNA of the rat beta-casein gene along with noncoding exon I and 0.5 kb of intron A were identified, most of which transmitted the transgenes to their offspring in a Mendelian pattern. CAT activity was detected predominantly in the lactating mammary gland of female transgenic mice but not in the male mammary fat pad. A several-hundred-fold variation in the level of cat expression was observed in the mammary gland of different lines of mice, presumably due to the site of integration of the transgenes. CAT activity was increased in the mammary gland during development from virgin to midpregnancy and lactation. Unexpectedly, the casein-cat transgenes were also expressed in the thymus of different lines of both male and female mice, in some cases at levels equivalent to those observed in the mammary gland, and in contrast to the mammary gland, CAT activity was decreased during pregnancy and lactation in the thymus. Thus, 0.5 kb of 5'-flanking DNA of the rat beta-casein gene along with noncoding exon I and 0.5 kb of intron A are sufficient to target bacterial cat gene expression to the mammary gland of lactating mice.

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