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Benefits of Functional Foods in Nutrient Imbalance of Vulnerable Populations

Posted in Chemical Biology and its relations to Metabolic Disease, Metabolomics, Nutrigenomics, Nutrition, Nutritional Supplements: Atherogenesis, lipid metabolism, Population Health Management, Nutrition and Phytochemistry, tagged Antioxidant, diet, Dr. Sudipta Saha, Essential nutrient, essential nutrients, food, food supplements, Functional food, functional foods, Nutraceutical, Nutrient, nutrient imbalance, Nutrition on March 24, 2013| 2 Comments »

Benefits of Functional Foods in Nutrient Imbalance of Vulnerable Populations

Reporter and Curator: Dr. Sudipta Saha, Ph.D.

There are clear distinctions between a food and a drug. Nutraceuticals, however, occupy a place between the two. Nutraceuticals are naturally derived phytochemicals with potential health benefits and without the characteristics of being essential nutrients. Foods that contain these non-essential substances with potential health benefits may qualify as “functional foods.” As defined by the Food and Nutrition Board of the National Academy of Sciences, the term functional food refers to foods that provide health benefits beyond basic nutrition. Examples of these are

psyllium seeds (soluble fiber),
soy foods (isoflavones),
cranberry juice (proanthocyanidins),
purple grape juice (resveratrol),
tomatoes (lycopene), and
green tea (catechins).

The bioactive components of functional foods:

flavonols,
monomeric and polymeric flavan-3-ols,
highly coloured anthocyanins, and
phenolic acids

may be increased in or added to traditional foods. An example is a genetically modified tomato high in lycopene, which has potent antioxidant capabilities.

The risk of nutrient imbalance is highest in vulnerable populations unable to access essential or conditionally essential nutrients. To a large extent, the

very young and the
frail elderly

are the select groups who might benefit most from alleviating this risk. The lack of adequate nutrition may be due to seasonal and unexpected losses of agricultural produce; however, poverty is a factor on a global scale as a result of growing economic disparities. The question then becomes what role functional foods offer to improve recognized population nutritional deficiencies. The range of work being done on functional foods is impressive, from

modified oils that contain heart-healthy ω-3 fatty acids to
cassava plants developed with an increased protein content to help counter malnutrition in developing nations.

However, the nutraceutical industry has responded to and relies on the untested expectations of the healthiest members of the world’s population rather than its more vulnerable ones. Due largely to economic causes, those in need are less likely to receive the benefits of nutraceuticals from whole foods or from manufactured foods or supplements. This is particularly striking where the source is locally available and extracted for commerce but is unaffordable or unavailable to the native population.

The rapid advances in biotechnology and functional foods confront us with a need to address the benefits of these with regard to improving health and managing or decreasing disease risks. Conventional dietary recommendations have focused on the consumption of fruits, vegetables, legumes, and whole grains, a decreased sugar intake, and an emphasis on plant oils, recommendations that have unproved benefits for the prevention of chronic diseases and that have complexities involving individual, environmental, and genetic influences.

Although the potential benefits of phytochemicals could have an impact on health status for vulnerable populations, the recommendations focused on plant foods do not address the primary concerns of the undernutrition associated with a poor quality of protein intake. Taken individually, plant sources do not provide a balanced amino acid profile necessary for protein synthesis, being deficient in lysine and/or methionine. Animal sources of protein, specifically meat and fish, also provide essential fatty acids not found in plant sources of protein and that may be otherwise limited. In addition, plants may contain antinutritional factors (wheat, cassava roots, cabbages, soy beans), and plant-based diets may be deficient in important essential nutrients.

Programs must focus on the sustainable production and local processing of indigenous products that can be used by needy populations to improve their nutritional intake and enhance economic stability. In addition, dietary recommendations must not exclude important sources of nutrition for more vulnerable populations by focusing primarily on plant-based sources of food, decreasing saturated fat, and de-emphasizing the importance of high-value biologic protein. The global economic crisis has touched the lives of 80% of the population in most developing countries with a threat to the development of a generation of children (approximately 250 million) who are most vulnerable in the first 2 years of life. An investment in nutrition in this circumstance has a high value, and the use of complementary food supplements to increase a meal’s nutrient content is warranted.

A recent proposal has concluded there are health benefits for foods and food constituents put together in a synergic diet pattern, suggesting that the interrelation between constituents within whole foods is significant, and has recommended dietary variety and the selection of nutrient-rich foods. Providing vulnerable populations with an adequate supply of whole foods should take precedence over the recommendation of food products in supplying not only essential macro- and micronutrients and energy but also phytochemicals whose value to the human diet is still to be determined.

Source References:

http://www.sciencedirect.com/science/article/pii/S0899900711003133

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Congenital Hyperinsulinism

Posted in Biological Networks, Gene Regulation and Evolution, Chemical Genetics, Genome Biology, Genomic Testing: Methodology for Diagnosis, Medical and Population Genetics, Nutrigenomics, Personalized and Precision Medicine & Genomic Research, Population Health Management, Genetics & Pharmaceutical, Reproductive Andrology, Embryology, Genomic Endocrinology, Preimplantation Genetic Diagnosis and Reproductive Genomics, tagged Beckwith–Wiedemann syndrome, beta cells, Conditions and Diseases, congenital, Congenital disorder, Congenital hyperinsulinism, Dr. Sudipta Saha, genetic disorder, hyperinsulinism, Hypoglycemia, islets, Pancreas on February 11, 2013| Leave a Comment »

Reporter and Curator: Dr. Sudipta Saha, Ph.D.

Congenital hyperinsulinism is a medical term referring to a variety of congenital disorders in which hypoglycemia is caused by excessive insulin secretion. Congenital forms of hyperinsulinemic hypoglycemia can be transient or persistent, mild or severe. These conditions are present at birth and most become apparent in early infancy. The severe forms can cause obvious problems in the first hour of life, but milder forms may not be detected until adult years. Mild cases can be treated by frequent feedings, more severe cases can be controlled by medications that reduce insulin secretion or effects, and a minority of the most severe cases require surgical removal of part or most of the pancreas to protect the brain from damage due to recurrent hypoglycemia.

Types of congenital hyperinsulinism:

1. Transient neonatal hyperinsulinism

2. Focal hyperinsulinism

Paternal SUR1 mutation with clonal loss of heterozygosity of 11p15
Paternal Kir6.2 mutation with clonal loss of heterozygosity of 11p15

3. Diffuse hyperinsulinism

a. Autosomal recessive forms

i. SUR1 mutations
ii. Kir6.2 mutations
iii. Congenital disorders of glycosylation

b. Autosomal dominant forms

i. Glucokinase gain-of-function mutations
ii. Hyperammonemic hyperinsulinism (glutamate dehydrogenase gain-of-function mutations)
iii. Short chain acyl coenzyme A dehydrogenase deficiency

4. Beckwith-Wiedemann syndrome (thought to be due to hyperinsulinism but pathophysiology still uncertain: 11p15 mutation or IGF2 excess)

Congenital hyperinsulinism (CHI or HI) is a condition leading to recurrent hypoglycemia due to an inappropriate insulin secretion by the pancreatic islet beta cells. HI has two main characteristics:

a high glucose requirement to correct hypoglycemia and
a responsiveness of hypoglycemia to exogenous glucagon.

HI is usually isolated but may be rarely part of a genetic syndrome (e.g. Beckwith-Wiedemann syndrome, Sotos syndrome etc.). The severity of HI is evaluated by the glucose administration rate required to maintain normal glycemia and the responsiveness to medical treatment. Neonatal onset HI is usually severe while late onset and syndromic HI are generally responsive to a medical treatment. Glycemia must be maintained within normal ranges to avoid brain damages, initially, with glucose administration and glucagon infusion then, once the diagnosis is set, with specific HI treatment. Oral diazoxide is a first line treatment.

In case of unresponsiveness to this treatment, somatostatin analogues and calcium antagonists may be added, and further investigations are required for the putative histological diagnosis:

pancreatic (18)F-fluoro-L-DOPA PET-CT and
molecular analysis.

Indeed, focal forms consist of a focal adenomatous hyperplasia of islet cells, and will be cured after a partial pancreatectomy.

Diffuse HI involves all the pancreatic beta cells of the whole pancreas. Diffuse HI resistant to medical treatment (octreotide, diazoxide, calcium antagonists and continuous feeding) may require subtotal pancreatectomy which post-operative outcome is unpredictable.

The genetics of focal islet-cells hyperplasia associates

a paternally inherited mutation of the ABCC8 or
the KCNJ11 genes, with
a loss of the maternal allele specifically in the hyperplasic islet cells.

The genetics of diffuse isolated HI is heterogeneous and may be

recessively inherited (ABCC8 and KCNJ11) or
dominantly inherited (ABCC8, KCNJ11, GCK, GLUD1, SLC16A1, HNF4A and HADH).

Syndromic HI are always diffuse form and the genetics depend on the syndrome. Except for HI due to

potassium channel defect (ABCC8 and KCNJ11),

most of these HI are sensitive to diazoxide.

The main points sum up the management of HI:

i) prevention of brain damages by normalizing glycemia and
ii) screening for focal HI as they may be definitively cured after a limited pancreatectomy.

Source & References:

http://en.wikipedia.org/wiki/Congenital_hyperinsulinism

http://www.ncbi.nlm.nih.gov/pubmed/20550977

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Genomic Endocrinology and its Future

Posted in Biological Networks, Gene Regulation and Evolution, Biomarkers & Medical Diagnostics, Cell Biology, Signaling & Cell Circuits, Chemical Biology and its relations to Metabolic Disease, Genome Biology, Genomic Testing: Methodology for Diagnosis, Personalized and Precision Medicine & Genomic Research, Pharmacogenomics, Population Health Management, Genetics & Pharmaceutical, Reproductive Andrology, Embryology, Genomic Endocrinology, Preimplantation Genetic Diagnosis and Reproductive Genomics, tagged disease, DNA, Dr. Sudipta Saha, endocrinology, Eukaryotic, gene, gene expression, genetic disorder, genetics, genome, genomics, hybridization on December 27, 2012| 3 Comments »

Reporter and Curator: Dr. Sudipta Saha, Ph.D.

A number of novel genes have been identified in association with a variety of endocrine phenotypes over the last few years. However, although mutations in a number of genes have been described in association with disorders such as

hypogonadotropic hypogonadism,
congenital hypopituitarism,
disorders of sex development, and
congenital hyperinsulinism,

these account for a minority of patients with these conditions, suggesting that many more genes remain to be identified.

How will these novel genes be identified? Monogenic disorders can arise as a result of genomic microdeletions or microduplications, or due to single point mutations that lead to a functional change in the relevant protein. Such disorders may also result from altered expression of a gene, and hence altered dosage of the protein. Candidate genes may be identified by utilizing naturally occurring or transgenic mouse models, and this approach has been particularly informative in the elucidation of the genetic basis of a number of disorders.

Other approaches include the identification of chromosomal rearrangements using conventional karyotyping techniques, as well as novel assays such as array comparative genomic hybridization (CGH) and single nucleotide polymorphism oligonucleotide arrays (SNP arrays). These molecular methods usually result in the identification of gross abnormalities as well as submicroscopic deletions and duplications, and eventually to the discovery of single gene defects that are associated with a particular phenotype.

However, there is no doubt that the major advances in novel gene identification will be made as a result of the sequencing of the genome of affected individuals and comparison with control data that are already available. Chip techniques allow hybridization of DNA or RNA to hundreds of thousands of probes simultaneously. Microarrays are being used for mutational analysis of human disease genes.

Complete sequencing of genomes or sequencing of exons that encode proteins (exome sequencing) is now possible, and will lead to the elucidation of the etiology of a number of human diseases in the next few years. High-throughput, high-density sequencing using microarray technology potentially offers the option of obtaining rapid, accurate, and relatively inexpensive sequence of large portions of the genome. One such technique is oligo-hybridization sequencing, which relies on the differential hybridization of target DNA to an array of oligonucleotide probes. This technique is ideally suited to the analysis of DNA from patients with defined disorders, such as disorders of sex development and retinal disease, but suffers from a relatively high false positive rate and failure to detect insertions and deletions.

It is often difficult to perform studies in humans, and so the generation of animal models may be valuable in understanding the etiology and pathogenesis of disease. A number of naturally occurring mouse models have led to the identification of corresponding candidate genes in humans, with mutations subsequently detected in human patients. More frequently, genes of interest are often deleted and lead to the generation of disease models.

In general, mouse models correlate well with human disease; however species-specific defects need to be taken into account. Additionally, the transgenic models could be used to manipulate a condition, with the potential for new therapies. The advent of conditional transgenesis has led to an exponential increase in our understanding of how the mutation of a single gene impacts on a single organ. Using technology such as inducible gene expression systems, the effect of switching on or switching off a gene at a particular stage in development can be determined.

Advances in genomics will also have a major impact on therapeutics. Micro RNAs (miRNA) are small non-coding RNAs that regulate gene expression by targeting mRNAs of protein coding genes or non-coding RNA transcripts. Micro RNAs also have an important role in developmental and physiological processes and can act as tumor suppressors or oncogenes in the ontogenesis of cancers. The use of small interfering RNA (siRNA) offers promise of novel therapies in a range of conditions, such as cystic fibrosis and Type II autosomal dominant IGHD. Elucidation of the genetic basis of disease also allows more direct targeting of therapy. For instance, children with permanent neonatal-onset diabetes mellitus (PNDM) due to mutations in SUR1 or KIR6.2 were previously treated with insulin but have now been shown to respond well to sulfonylureas, thereby allowing the cessation of insulin therapy.

Finally, we are now entering the era of pharmacogenetics when the response of an individual to various therapeutic agents may be determined by their genotype. For example, a polymorphism in the GH receptor that results in deletion of exon 3 may be associated with an improved response to GH. Thus the elucidation of the genetic basis of many disorders will aid their management, and permit the tailoring of therapy in individual patients.

Source References:

http://www.frontiersin.org/Genomic_Endocrinology/10.3389/fendo.2011.00011/full

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Genetics and male endocrinology

Posted in Biological Networks, Gene Regulation and Evolution, Genome Biology, Genomic Testing: Methodology for Diagnosis, Molecular Genetics & Pharmaceutical, Personalized and Precision Medicine & Genomic Research, Population Health Management, Genetics & Pharmaceutical, Reproductive Andrology, Embryology, Genomic Endocrinology, Preimplantation Genetic Diagnosis and Reproductive Genomics, tagged Androgen insensitivity syndrome, androgen receptor, Dr. Sudipta Saha, endocrinology, genetics, ligand, phenotype on November 12, 2012| 2 Comments »

Reporter and Curator: Dr. Sudipta Saha, Ph.D.

Genetics and Male Endocrinology

Image Source: Created by Noam Steiner Tomer 8/10/2020

Male sexual differentiation and development proceed under direct control of androgens. Androgen action is mediated by the intracellular androgen receptor, which belongs to the superfamily of ligand-dependent transcription factors. Mutations in the androgen receptor gene cause phenotypic abnormalities of male sexual development that range from a:

female phenotype (complete testicular feminization), to that of
under-virilized or infertile men.

Using the tools of molecular biology, it was analyzed androgen receptor gene mutations in 31 unrelated subjects with androgen resistance syndromes. Most of the defects are due to nucleotide changes that cause premature termination codons or single amino acid substitutions within the open reading frame encoding the androgen receptor, and the majority of these substitutions are localized in three regions of the androgen receptor:

the DNA-binding domain and
two segments of the androgen-binding domain.

Less frequently, partial or complete gene deletions have been identified. Functional studies and immunoblot assays of the androgen receptors in patients with androgen resistance indicate that in most cases the phenotypic abnormalities are the result of impairment of receptor function or decreases in receptor abundance or both.

In the X-linked androgen insensitivity syndrome, defects in the androgen receptor gene have prevented the normal development of both internal and external male structures in 46, XY individuals.

The complete form of androgen insensitivity syndrome is characterized by

46, XY karyotype,
external female phenotype,
intra-abdominal testes,
absence of uterus and ovaries,
blindly ending vagina, and
gynecomastia.

There is also a group of disorders of androgen action that result from partial impairment of androgen receptor function. Clinical indications can be abnormal sexual development of individuals with a

predominant male phenotype with
severe hypospadias and micropenis or of individuals with a
predominantly female phenotype with cliteromegaly,
ambiguous genitalia, and
gynecomastia.

Complete or gross deletions of the androgen receptor gene have not been frequently found in persons with the complete androgen insensitivity syndrome, whereas point mutations at several different sites in exons 2-8 encoding the DNA- and androgen-binding domain have been reported in both partial and complete forms of androgen insensitivity, with a relatively high number of mutations in two clusters in exons 5 and 7.

The number of mutations in exon 1 is extremely low, and no mutations have been reported in the hinge region, located between the DNA-binding domain and the ligand-binding domain.

The X-linked condition of spinal and bulbar muscle atrophy (Kennedy’s disease) is characterized by a progressive motor neuron degeneration associated with signs of androgen insensitivity and infertility. The molecular cause of spinal and bulbar muscle atrophy is an expanded length (> 40 residues) of one of the polyglutamine stretches in the N-terminal domain of the androgen receptor.

Source References:

http://www.ncbi.nlm.nih.gov/pubmed/8421085

http://www.ncbi.nlm.nih.gov/pubmed/8732995

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Leptin and Puberty

Posted in Population Health Management, Genetics & Pharmaceutical, Reproductive Andrology, Embryology, Genomic Endocrinology, Preimplantation Genetic Diagnosis and Reproductive Genomics, Reproductive Biology & Bio Instrumentation, tagged Adipose tissue, Dr. Sudipta Saha, GH, GnRH, leptin, LH, Messenger RNA, puberty on November 5, 2012| 5 Comments »

Reporter and Curator: Dr. Sudipta Saha, Ph.D.

It is well established that food restriction delays pubertal onset, whereas refeeding abolishes this delay. In addition, murine and human genetic models of leptin deficiency fail to enter puberty, and treatment with leptin can establish a pulsatile secretory pattern of gonadotropins that is characteristic of early puberty. The female transgenic skinny mouse, which is an in vivo model of chronic hyperleptinemia in the absence of adipose tissue, enters puberty precociously. Data regarding the effects of leptin administration on pubertal onset are controversial. It has been shown that intracerebroventricular leptin administration prevents the delay in vaginal opening induced by chronic food restriction in the rat. By contrast, it has been found that artificially raised leptin levels are not sufficient to abolish the delay of pubertal onset caused by food deprivation. Thus, the question arises whether leptin might be a ‘permissive factor’ (tonic mediator), whose concentration above a certain threshold is required for pubertal onset, or a ‘trigger’ (phasic mediator) that determines the pubertal spurt through a rise in serum concentration at an appropriate time of development.

The temporal correlation between increases in leptin concentration and the initiation of LH pulsatility over the peripubertal period has been studied in several species. In men it has been shown that leptin levels rise by 50% before the onset of puberty, and decrease to baseline after the initiation of puberty. Other cross-sectional studies showed that age has a significant effect on serum leptin concentrations through prepuberty into early puberty. It has been reported repeatedly that there are no significant changes in leptin levels over the peripubertal period in male rhesus macaques; however, more recent studies performed in castrated male monkeys showed that nocturnal levels of leptin increase just before the nocturnal prepubertal increase in pulsatile LH release.

A possible explanation for such contrasting reports in monkeys could be the sampling of nocturnal rather than diurnal blood. Indeed, in primates, prepubertal changes in nocturnal LH release occur approximately five months before diurnal variations. Another reason might be the use of different models: agonadal monkeys were treated with intermittent exogenous GnRH to sensitize the pituitary to endogenous GnRH, thus magnifying the LH release independently from gonadal influences. In the same study, the leptin rise was accompanied by a sustained increase in nocturnal GH and IGF-I concentrations before the onset of puberty, which is defined as the increase in nocturnal pulsatile LH secretion. It is not clear whether one of the two metabolic signals has a predominant role or whether both act in concert. Indeed, it has been reported that the maximum increase in GH and leptin occurs simultaneously, about 10–30 days before the onset of puberty. However, these conclusions were based on results from a study that used castrated animals, which in the strictest sense do not undergo puberty. Thus, it remains to be clarified whether the same mechanisms that result in the onset of the pubertal rise in LH secretion in castrated animals are also responsible for the reactivation of the HPG axis in intact animals.

The sexual dimorphism in leptin concentrations becomes evident after puberty. In males, leptin levels rise throughout childhood, reach a peak in the early stages of puberty and then decline, whereas they increase steadily during pubertal development in females. Consequently, leptin levels are three to four times higher in females than in males. The reason for this postpubertal sexual dimorphism in leptin levels is not clear. After puberty, serum testosterone and testicular volume are inversely related to leptin levels in males, whereas in females, when adjusted for adiposity indexes, estradiol is directly correlated with leptin levels. These observations indicate that androgens and estradiol might account, at least in part, for the gender differences in circulating leptin levels. This is also supported by in vitro studies which show that androgens and estrogens inhibit and stimulate leptin expression and release from human adipocytes in culture, respectively.

Thus, puberty represents a turning point in the sexual dimorphic relationships between the HPG axis and leptin by determining the steroid milieu that leads to a different regulation of leptin secretion in the sexes.

Source References:

http://www.sciencedirect.com/science/article/pii/S1043276000003520#

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Gene Trap Mutagenesis in Reproductive Research

Posted in Bio Instrumentation in Experimental Life Sciences Research, Biological Networks, Gene Regulation and Evolution, Biomarkers & Medical Diagnostics, Cell Biology, Signaling & Cell Circuits, Chemical Genetics, Computational Biology/Systems and Bioinformatics, Disease Biology, Small Molecules in Development of Therapeutic Drugs, Genome Biology, Genomic Testing: Methodology for Diagnosis, Medical and Population Genetics, Molecular Genetics & Pharmaceutical, Personalized and Precision Medicine & Genomic Research, Pharmaceutical Analytics, Pharmaceutical Industry Competitive Intelligence, Pharmaceutical R&D Investment, Population Health Management, Genetics & Pharmaceutical, Proteomics, Reproductive Andrology, Embryology, Genomic Endocrinology, Preimplantation Genetic Diagnosis and Reproductive Genomics, Reproductive Biology & Bio Instrumentation, Stem Cells for Regenerative Medicine, Technology Transfer: Biotech and Pharmaceutical, tagged DNA, Dr. Sudipta Saha, gene, gene trap mutagenesis, genetics, genome, microarray, mutation, Nucleic acid sequence, reproductive research, Single-nucleotide polymorphism on October 18, 2012| 3 Comments »

Reporter and Curator: Dr. Sudipta Saha, Ph.D.

With the completion of the mapping of the human genome, we now have access to all the DNA sequence information responsible for human biology. Together with microarray technology, we are ushering in a new era in reproductive medicine—the era of Reproductive Genomics.

Whole genome microarray analysis of the testis and ovary suggests that a substantial part of the genome is expressed in reproductive tissues and many of them are likely to be important for normal reproduction. Yet adequate expression and functional information is only available for less than 10% of them. Hence, one of the important questions in reproductive studies now is ‘how do we associate function with the genes expressed in reproductive tissues?’ The establishment of mutations in animal models such as the mouse represents one powerful approach to address this question.

Animal models have played critical roles in improving our understanding of mechanisms and pathogenesis of diseases. Mouse knockout models have often provided highly needed functional validation of genes implicated in human diseases. The rapid advance of human genetics in areas such as

single nucleotide polymorphisms (SNP) and
haplotyping technology

now allows the identification of disease-associated single nucleotide variation at a much faster pace. Functional examination of those candidate genes is needed to determine if those genes or variants are indeed involved in reproductive disease. Generating mutations in murine homologs of candidate genes represents a direct way to determine their roles, and mouse models will further allow the dissection of genetic pathways underlying the disease condition and provide models to test possible drug treatments. Thus, how to generate mouse models efficiently becomes a priority issue in the Genomics era of Reproductive Medicine.

It is known that generating a mouse knockout is no small endeavor, even for a mouse research lab, often requiring specialized expertise and experience in

molecular biology,
embryonic stem (ES) biology and
mouse husbandry.

Therefore, it could be intimidating for people who have little experience in mouse research. Fortunately, there are some technological developments in the mouse community that make the task of generating mouse mutations less intimidating to people unfamiliar with mouse genetics. One of these developments is the effort led by the International Gene Trap Consortium (IGTC) to generate a library of mouse mutant ES cells covering most of the genes in the mouse genome. This method saves researchers the tedious and sometimes challenging tasks of making knockout vectors and screening ES cell colonies and directly provides researchers an ES cell clone carrying the mutation of the gene of interest.

Because gene trapping involves the use of different mechanisms in generating mutations from the traditional knockout method, and its efficacy in targeting reproductive genes which often are expressed in later development or adult has not been fully established, it is necessary to examine the benefits and limitations of this technology, especially in the perspective of reproductive medicine so that reproductive researchers and physicians who are interested in mouse models could become familiar with this technology.

With this in mind, we provide an overview of the gene trapping mutagenesis method and its possible application to Reproductive Medicine. We evaluate gene trapping as a method in terms of its efficiency in comparison with traditional knockout methods and use an in-house software program to screen the IGTC database for existing cell lines with possible mutations in genes expressed in various reproductive tissues. Among over seven thousand genes highly expressed in human ovaries, almost half of them have existing gene trap lines.

Additionally, from 900 human seminal fluid proteins, 43% of them have gene trap hits in their mouse homologs. Our analysis suggests gene trapping is an effective mutagenesis method for identifying the genetic basis of reproductive diseases and many mutations for important reproductive genes are already present in the database. Given the rapid growth of the number of gene trap lines, the continuing evolution of gene trap vectors, and its easy accessibility to scientific communities, gene trapping could provide a fast and efficient way of generating mouse mutation(s) for any one particular gene of interest or multiple genes involved in a pathway at the same time. Consequently, we recommend gene trapping to be considered in the planning of mouse modeling of human reproductive disease and the IGTC be the first stop for people interested in searching for and generating mouse mutations of genes of interest.

Gene trapping is a high-throughput approach of generating mutations in murine ES cells through vectors that simultaneously disrupt and report the expression of the endogenous gene at the point of insertion. First-generation vectors trapped genes that were actively transcribed in undifferentiated ES cells. Depending on the areas in which they integrate, these vectors can be roughly divided into two classes:

promoter trap vectors and
gene trap vectors.

Promoter trap vectors contain promoterless reporter regions, usually bgeo (a fusion of neomycin phosphotransferase and b-galactosidase), and thus have to be integrated into an exon of a transcriptionally active locus in order for the cell to be selected for neomycin resistance or by LacZ staining. Gene trap vectors demonstrate more utility by their added ability to integrate into an intron. These vectors contain a splice acceptor (SA) site positioned at the 50-end of the reporter gene, allowing the vector to be spliced to the endogenous gene to form a fusion transcript. Later improvements include an internal ribosomal re-entry site (IRES) between the SA site and the reporter gene sequence; as a result, the reporter gene can be translated even when it is not fused to the trapped gene. Second-generation vectors have sought to trap genes that are transcriptionally silent in ES cells. Although these vectors still contain a promoterless reporter gene with a 50 SA sequence, the antibiotic resistance gene is under the control of a constitutive promoter. Consequently, antibiotic selection is independent from the expression of the trapped gene, whereas the expression of the reporter gene is still regulated by the endogenous promoter.

A disadvantage of these vectors is that all integration events give rise to resistant ES cells regardless of whether or not the vector has integrated into a gene locus. To increase trapping efficiency, a new class of polyA gene trap vectors was developed where the polyadenylation signal of the neo gene was replaced by a splice donor sequence, thereby requiring the vector to trap an endogenous polyA signal for expression of neo. These vectors were recently shown to have a bias toward insertion near the 30-end of a gene due to nonsense-mediated mRNA decay of the fusion transcript. An improved polyA trap vector, UPATrap, was developed to overcome this bias using an IRES sequence placed downstream of a marker containing a termination codon. Gene trap vectors are usually introduced by retroviral infection or electroporation of plasmid DNA, with each approach having its own advantages and disadvantages.

While relatively difficult to manipulate, retroviral gene traps display a preference toward insertion at the 50-end of genes, which is advantageous for generating null alleles. Moreover, the multiplicity of infection with retroviruses can be tightly controlled to a single trap event or simultaneous disruption in many genes. However, there may be a possible bias integration toward certain ‘hotspots’ of the genome.

In contrast, plasmid-based gene trap vectors integrate more randomly into the genome. This can, however, potentially result in a functional partial protein and a hypomorphic phenotype. Additionally, plasmid vectors usually result in multiple integrations in 20–50% of cell lines. The most common approach for identifying the gene trap integration site is to use 50 or 30 rapid amplification of cDNA ends (RACE) to amplify the fusion transcript. The sequence provides a DNA tag for the identification of the disrupted gene and can be used for genotypic screens. Mutagenesis screens can also be performed on the basis of gene function or expression, and data from an expression sequence combined with sequence tag information can elucidate novel expression patterns of known genes or to suggest gene function.

Gene trapping has proven to be an efficacious technique in mutagenesis compared with other methods such as

spontaneous mutations,
fortuitous transgene integration and
N-ethyl-N-nitrosurea (ENU) mutagenesis

We have been able to use our SpiderGene program to identify genes in reproductive tissues that are present in the IGTC database and moreover to narrow down those with restricted expression in the testis and ovary. Gene trapping possesses an enormous potential for researchers in the reproductive field seeking to create mouse models for a gene mutation. The improving versatility of gene trap vectors has enabled groups to trap an increasing number of genes in various organisms, including Arabidopsis, Zebra fish and Drosophila.

The gene trap effort has perhaps been the most extensive in the murine genome, with over 57000 cell lines representing more than 40% of the known genome. These large-scale screens will likely achieve the trapping of the entire mouse genome in the coming years, but the power of gene trapping will only be fully demonstrated by its usefulness in investigator-driven focused functional analyses.

In our laboratory, future work will focus on generating knockout mice in order to investigate gene function and to identify gene products that might have therapeutic value in reproduction. As screening efforts continue, gene trapping will continue to be a valuable tool in mouse genomics and will undoubtedly yield new discoveries in Reproductive Physiology and Pathology.

Source References:

http://www.ncbi.nlm.nih.gov/pubmed?term=Gene%20trap%20mutagenesis%3A%20a%20functional%20genomics%20approach%20towards%20reproductive%20research