THE ALTERATION OF MILK CONTENT BY GENETIC ENGINEERING AND RECOMBINANT DNA-MEDIATED SELECTION TECHNIQUES

Since the advent of gene cloning technology, suggestions have been made of specific alterations that could be engineered into the genomes of cattle, sheep or goats to alter their milk composition. These alterations could affect milk protein characteristics or quantity in ways which might be of benefit to milk processing and dairy product manufacturing industries. Other changes could improve the suitability of bovine milk for human consumption. In addition, animals could be engineered to synthesize valuable foreign proteins, such as pharmaceuticals, to be secreted in milk. In this paper, we evaluate these suggestions critically, taking into account current understanding of milk composition and processing, as well as recent information concerning the structure and regulation of genes coding for proteins involved in determining milk content. All these suggested alterations depend on successful production of transgenic animals capable of efficiently expressing introduced genes. The view is examined that some improvements in type or amount of certain milk proteins might better be achieved by conventional breeding practices, or by breeding systems that would rely on recombinant DNA techniques for methods of selection. Key words: Casein, beta-lactoglobulin, α-lactalbumin, plasmin, transgenic, bovine, ovine, lactose

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Expression of bovine β-lactoglobulin transgenic mice

Journal of Dairy Research ◽

10.1017/s0022029999003428 ◽

1999 ◽

Vol 66 (2) ◽

pp. 289-294 ◽

Cited By ~ 4

Author(s):

ALFONSO GUTIÉRREZ-ADÁN ◽

ELIZABETH A. MAGA ◽

ESMAIL BEHBOODI ◽

JANICE S. CONRAD-BRINK ◽

ANTHONY G. MACKINLAY ◽

...

Keyword(s):

Mammary Gland ◽

Transgenic Mice ◽

Transgenic Animals ◽

Milk Composition ◽

Developmentally Regulated ◽

Independent Manner ◽

Milk Protein Genes ◽

Dairy Animals ◽

Foreign Proteins ◽

Β Lactoglobulin

The use of transgenic animals to manipulate milk composition has considerable potential, both for the production of biomedical proteins and for the direct manipulation of milk composition for the improvement of dairy animals and their products (for reviews, see Wall et al. 1992; Yom & Bremel, 1993). Promoters from a number of milk protein genes from a variety of species have been tested for their ability to direct the expression of foreign proteins to the mammary gland (for review, see Maga & Murray, 1995).β-Lactoglobulin (β-lg) is the major whey protein produced in ruminant milk and is part of the normal milk composition of most mammals except humans and rodents (Pervaiz & Brew, 1985). It is expressed at high levels in the mammary gland and is developmentally regulated. Transgenic mice have been produced using the complete ovine (Simons et al. 1987; Shani et al. 1992) and caprine (Ibañez et al. 1997) β-lg genes. In general, high levels of expression were obtained with the ovine β-lg gene, and expression was also seen in a position-independent manner (Whitelaw et al. 1992). Lower levels of expression were reported using the caprine β-lg gene. Here we report the production of transgenic mice using the bovine β-lg gene. We describe high expression, position-dependent, and copy number-related expression of bovine β-lg protein in the milk of six lines of transgenic mice.

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A hypomorphic mutation in the mouse Csn1s1 gene generated by CRISPR/Cas9 pronuclear microinjection

Vavilov Journal of Genetics and Breeding ◽

10.18699/vj21.036 ◽

2021 ◽

Vol 25 (3) ◽

pp. 331-336

Author(s):

A. V. Smirnov ◽

T. A. Shnaider ◽

A. N. Korablev ◽

A. M. Yunusova ◽

I. A. Serova ◽

...

Keyword(s):

Gene Expression ◽

Gene Knockout ◽

Milk Proteins ◽

Transgenic Animals ◽

Regulatory Elements ◽

Milk Composition ◽

Peptide Sequence ◽

Start Codon ◽

Signal Peptide Sequence ◽

Casein Genes

Caseins are major milk proteins that have an evolutionarily conserved role in nutrition. Sequence variations in the casein genes affect milk composition in livestock species. Regulatory elements of the casein genes could be used to direct the expression of desired transgenes into the milk of transgenic animals. Dozens of casein alleles have been identified for goats, cows, sheep, camels and horses, and these sequence variants are associated with altered gene expression and milk protein content. Most of the known mutations affecting casein genes’ expression are located in the promoter and 3’-untranslated regions. We performed pronuclear microinjections with Cas9 mRNA and sgRNA against the first coding exon of the mouse Csn1s1 gene to introduce random mutations in the α-casein (Csn1s1) signal peptide sequence at the beginning of the mouse gene. Sanger sequencing of the founder mice identified 40 mutations. As expected, mutations clustered around the sgRNA cut site (3 bp from PAM). Most of the mutations represented small deletions (1–10 bp), but we detected several larger deletions as well (100–300 bp). Functionally most mutations led to gene knockout due to a frameshift or a start codon loss. Some of the mutations represented in-frame indels in the first coding exon. Of these, we describe a novel hypomorphic Csn1s1 (Csn1s1c.4-5insTCC) allele. We measured Csn1s1 protein levels and confirmed that the mutation has a negative effect on milk composition, which shows a 50 % reduction in gene expression and a 40–80 % decrease in Csn1s1 protein amount, compared to the wild-type allele. We assumed that mutation affected transcript stability or splicing by an unknown mechanism. This mutation can potentially serve as a genetic marker for low Csn1s1 expression.

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Effect of polymorphisms in the leptin, leptin receptor and acyl-CoA:diacylglycerol acyltransferase 1 (DGAT1) genes and genetic polymorphism of milk proteins on bovine milk composition

Journal of Dairy Research ◽

10.1017/s0022029911000859 ◽

2011 ◽

Vol 79 (1) ◽

pp. 110-118 ◽

Cited By ~ 8

Author(s):

Maria Glantz ◽

Helena Lindmark Månsson ◽

Hans Stålhammar ◽

Marie Paulsson

Keyword(s):

Genetic Polymorphism ◽

Milk Protein ◽

Leptin Receptor ◽

Bovine Milk ◽

Genetic Selection ◽

Milk Proteins ◽

Milk Composition ◽

Milk Samples ◽

The Past ◽

Β Lactoglobulin

The relations between cow genetics and milk composition have gained a lot of attention during the past years, however, generally only a few compositional traits have been examined. The aim of this study was to determine if polymorphisms in the leptin (LEP), leptin receptor (LEPR) and acyl-CoA:diacylglycerol acyltransferase 1 (DGAT1) genes as well as genetic polymorphism of β-casein (β-CN), κ-CN and β-lactoglobulin (β-LG) impact several bovine milk composition traits. Individual milk samples from the Swedish Red and Swedish Holstein breeds were analyzed for components in the protein, lipid, carbohydrate and mineral profiles. Cow alleles were determined on the following SNP: A1457G, A252T, A59V and C963T on the LEP gene, T945M on the LEPR gene and Nt984+8(A-G) on the DGAT1 gene. Additionally, genetic variants of β-CN, κ-CN and β-LG were determined. For both the breeds, the same tendency of minor allele frequency was found for all SNPs and protein genes, except on LEPA1457G and LEPC963T. This study indicated significant (P<0·05) associations between the studied SNPs and several compositional parameters. Protein content was influenced by LEPA1457G (G>A) and LEPC963T (T>C), whereas total Ca, ionic Ca concentration and milk pH were affected by LEPA1457G, LEPA59V, LEPC963T and LEPRT945M. However, yields of milk, protein, CN, lactose, total Ca and P were mainly affected by β-CN (A2>A1) and κ-CN (A>B>E). β-LG was mainly associated with whey protein yield and ionic Ca concentration (A>B). Thus, this study shows possibilities of using these polymorphisms as markers within genetic selection programs to improve and adjust several compositional parameters.

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Role of Milk-Derived Antibacterial Peptides in Modern Food Biotechnology: Their Synthesis, Applications and Future Perspectives

Biomolecules ◽

10.3390/biom8040110 ◽

2018 ◽

Vol 8 (4) ◽

pp. 110 ◽

Cited By ~ 11

Author(s):

Muhammad Usman Khan ◽

Maryam Pirzadeh ◽

Carola Yvette Förster ◽

Sergey Shityakov ◽

Mohammad Ali Shariati

Keyword(s):

Side Effects ◽

Recombinant Dna ◽

Milk Proteins ◽

Living Organism ◽

Positive Influence ◽

Antibacterial Peptides ◽

Future Perspectives ◽

Food Biotechnology ◽

Recombinant Dna Techniques

Milk-derived antibacterial peptides (ABPs) are protein fragments with a positive influence on the functions and conditions of a living organism. Milk-derived ABPs have several useful properties important for human health, comprising a significant antibacterial effect against various pathogens, but contain toxic side-effects. These compounds are mainly produced from milk proteins via fermentation and protein hydrolysis. However, they can also be produced using recombinant DNA techniques or organic synthesis. This review describes the role of milk-derived ABPs in modern food biotechnology with an emphasis on their synthesis and applications. Additionally, we also discuss the mechanisms of action and the main bioproperties of ABPs. Finally, we explore future perspectives for improving ABP physicochemical properties and diminishing their toxic side-effects.

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Investigation of the milk composition of three Tsigai sheep varieties, and the detection of cazein fractions of sheep milk by Reversed-Phase HPLC

Acta Agraria Debreceniensis ◽

10.34101/actaagrar/31/2999 ◽

2008 ◽

pp. 13-22

Author(s):

Ildikó Bajúsz

Keyword(s):

Bovine Milk ◽

Milk Proteins ◽

Milk Composition ◽

Reversed Phase ◽

Hplc Method ◽

Solid Content ◽

Scientific Article ◽

Sheep Milk ◽

Milk Coagulation ◽

Primary Mission

The measurements started in 2005, the author used the milk of 3 types of Tsigai Sheep that are suitable for milking, they examined the milk composition, the possible differences between the milk types, the coagulation characteristics, cheese processing, yielding characteristics and the transfer of solid content into cheese. From the large number of measurements, the results of milk composition examinations are described.During the research the author examined the milk coagulation property of three types of Tsigai Sheep QTS 25 is a substance investigating instrument, and its primary mission is the analysis of the substance profile. The advantage of this instrument is the representation of the survey graphs from which the part of the substance characteristics could be directly read off. By the help of the applied mathematical and statistical operations we tried to establish connection between the congealment-hardness and the specialty of the different types of Tsigai Sheep.In 2006 the author began the identification and qualification of the major sheep milk proteins by RP- HPLC method. She used a scientific article written by Bordin et al. The method is able to separate and quantify the seven major proteins. However Bordin’s procedure was developed using bovine milk, the author adapted it to sheep milk.

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Antigenicity and Safety Evaluation of Lactiplantibacillus plantarum 7-2 Screened to Reduce α-Casein Antigen

Foods ◽

10.3390/foods11010088 ◽

2021 ◽

Vol 11 (1) ◽

pp. 88

Author(s):

Guangqing Mu ◽

Zhao Zhang ◽

Jiayi Wang ◽

Shujuan Jiang ◽

Hongxin Wang ◽

...

Keyword(s):

Milk Protein ◽

Dairy Product ◽

Bovine Milk ◽

Safety Evaluation ◽

Milk Proteins ◽

Enzyme Linked Immunosorbent Assay ◽

Sds Page ◽

Selection For ◽

The Ideal

α-Casein (α-CN) is considered the main allergen in bovine milk. Lactic acid bacteria (LAB) fermentation can hydrolyze milk protein and therefore reduce the antigenicity. In this paper, a LAB reducing the antigenicity of casein, identified as LactiplantibacillusPlantarum 7-2 (L. plantarum 7-2), was primarily identified by screening for protein hydrolysis ability using a method involving the determination of released free amino acid with further selection for the ideal antigenicity-reducing capability by enzyme-linked immunosorbent assay (ELISA). In order to verify the capability of L. plantarum 7-2 in inhibiting antigenicity, the standard milk proteins α-LA, β-LG, α-CN, β-CN and κ-CN were cultured with L. plantarum 7-2 for 18 h; The results of SDS-PAGE show that all the bands corresponding to the full length tested proteins became unclear or completely disappeared indicating that these proteins were hydrolyzed by L. plantarum 7-2. Correspondingly, the antigenicities of α-CN and β-LG were significantly reduced. L. plantarum 7-2 demonstrated negative hemolysis and nitrate reductase capabilities and was sensitive to the commonly used antibiotics ampicillin clindamycin tetracycline chloramphenicol, and erythromycin, demonstrating that L. plantarum 7-2 could be used in dairy product fermentation to reduce the antigenicity of milk protein.

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