Sperm Mediated Gene Transfer in Mammals; a Versatile Platform with Multiple Enhancements Techniques

In the field of animal transgenesis, many attempts have been made recently to simplify facilitate, and reduce the cost and labor required to do such tasks. Although several transgenesis techniques (such as DNA microinjection and somatic cell nuclear transfer) have been applied successfully to produce transgenic animals, these traditional techniques are so tedious and have several disadvantages. Retroviral mediated gene transfer has solved some of these usual problems but has, however, inevitable disadvantages represented most prominently by its biological hazard. Many researchers found that the most simple and non-cost effective way to produce transgenic animals is to focus on the natural ability of the sperm to “carry” the foreign DNA and to “fertilize” the oocyte. The most important breakthrough obtained in this aspect is the accumulated information that demonstrated the ability of foreign DNA to be internalized into the sperm head after simple incubation step. Accordingly, the only manipulation step is restricted into the head of the sperm. Then, nature will be allowed to fulfill its scheduled task of reproduction. This method known as sperm mediated gene transfer or SMGT. However, simple incubation of naked DNA with sperm head is not efficient enough to integrate the foreign DNA into the genome of the sperm. Thus, this review aims to pave the way for every effort to enable the researchers to undergo the transgenesis experiments in the routine laboratories. This is potentially can be done by testing the validity of the most modern enhancement approaches suggested on the original SMGT

Low Cost Alternative Method to Monitor the Electrophoretic Bands Substitution of the Positively Charged Rabbits Seminal Fluids

This paper aimed to evaluate the significance of a non-cost effective double dye technique in visualizing any DNA-seminal proteins interactions. After collecting rabbit’s ejaculate and removing sperm cells, ion exchange chromatography was performed to separate seminal proteins on the basis of their charge. Positively charged seminal proteins were eluted, lyophilized, and involved in this study. After incubating this eluted group with a standard reporter DNA, band shift assay was applied on this group. The results were compared. According to this study, we demonstrated the necessity of utilizing a double staining technique for the same band shift gel in order to ensure weather real or false band substitutions were obtained. Keywords : Rabbits seminal fluids ,DNA , Electrophoresi

The Efficiency of Transgenesis by Restriction Enzyme Mediated Integration - Sperm Mediated Gene Transfer (REMI-SMGT)

The present study tends to  test the validity of REMI-SMGT and to evaluate the efficiency of REMI-SMGT on generating other mammalian species rather than the sheep that made only by one group and to observe the possibility of doing so by using rabbits as a model for this approach and also to reduce the cost of REMI-SMGT by substituting liposomes and highly cost effective media with a high efficient, non-cost effective substitute. Direct protective relationship of liposome with DNA and seminal fluid was identified compared with DMSO.  While different treatments (linearized DNA – restriction enzyme – liposome complex, DNA – restriction enzyme – DMSO complex, DNA –DMSO complex, DNA –liposome complex, and even naked DNA) were all found to be successful to internalize inside the head of the sperm according to PCR results, only three (one by restriction enzyme – liposome treatment and two by restriction enzyme – DMSO treatment) out of fourteen new born babies were found to be transgenic by PCR. Despite the absolute ability of exogenous DNA to be internalized inside rabbit’s sperm head only few percent of transgenic babies were obtained. This may not reflect the weakness of restriction enzyme mediated transgenesis technique itself but it reflects the inability of recombinant sperm to fertilize superovulated oocyte compared with their normal counterparts. Comparable results were found between liposome and DMSO treatment which may reflect direct relationship of DMSO with the cell membrane instead of with the exogenous DNA itself as what is found with liposome. Key words : REMI-SMGT , PCR , DNA – restriction enzyme , DMS

Ion Exchange Fractionation of Rabbits Seminal Fluid: Recognizing a DNA Retardation Activity from the Main DNase Activity

The role of seminal proteins charge on the nature of seminal fluid inhibitory effect that exerted against exogenous DNA. Has been identified and an approach closely with more details to the nature of inhibitory activities available in rabbit seminal fluid proteins that prevent the entry of exogenous DNA into the head of sperm. After collection of rabbit’s ejaculate and removing sperm cells, seminal fluid was incubated with fixed concentration of exogenous DNA. The seminal fluid – exogenous DNA mixture was analyzed by electrophoresis. Ion exchange chromatography was used to separate seminal proteins on the basis of their charge. Positively charged proteins were eluted, lyophilized, and their profile was characterized by SDS-PAGE and native-PAGE. After incubation of this eluted group with the same source of DNA, the same electrophoretic conditions were applied on this group. According to our knowledge, this is the first paper in which ion exchange chromatography was used to separate two DNA counterfeiting activities of the seminal fluid using non-radioactive method in rabbits and even in other mammals. Thus, more than one inhibitory activity were identified and separated. DNA retardation activity (or DNA binding activity) which repressed DNA electrophoretic migration was the only activity that found to be available on the positively charged fractions while the DNase activity was found exclusively on the negatively charged group. Key words : Seminal fluid , transgenesis techniques , DNA electrophoretic migration , DNas

Different Factors Effects In Lactating Mother's Milk Compositions

This study was carried to estimate the effects of different factors on the total protein and lipid concentrations in lactating mother's milk, such as; home, age, body mass index, lactating stage and negative smoking habitat. Total Protein concentration and lipid concentration were estimated using Bradford method and Gravimetric method respectively. Results showed that lipid concentration(L.C) increased with age,  BMI, lactation stage, and affected by  negative smoking and increased in rural,  while protein concentration (P.C)was less effected by age, and decreased in obese and in negative smoking mothers, also it decreased in urban mothers. Protein profile showed slight differences between nonsmoking and negative smoking milk using SDS-PAGE electrophoresis. Key words: negative smoking, BMI, lactating mother’s milk.

Shortened Telomere Length in White Blood Cells of Patients with Non-Insulin Dependent Diabetes Mellitus (NIDDM)

Type 2 diabetes(TIIDM) aging-related disorder, is caused by a combination of peripheral insulin resistance and ?-cell dysfunction .Recent evidence, however, suggests that TIIDM is additionally characterized by impaired ?-cell regeneration and reduced ?-cell mass .Shortened telomeres have been previously associated with diabetes in several small-scale studies(Zhu et al.,2011). Measurement of telomere length in diabetic patients in different age and different duration of disease and compared with control. Also included the comparison telomere length between male and female for both control and diabetic groups and  between patients from genetic origin (mother origin or father origin) and patients from non genetic origin  . Genomic DNA was prepared from whole blood extraction using genaid kit and was quantified by Nanodrop .Terminal restriction fragment (TRF) lengths were measured using the Southern-blotting technique .This study was conducted between November 2010- November 2012 and, it was carried out at the diabetic Centre / Merjan Teaching Hospital in Babel Province by taking 54 diabetic patients(Type II DM) with disease duration (0-5),(>5-10 )and (>10 )years ,with age average (35-65 year)and most of them were on oral hypoglycemic drugs. While the study included 18 people apparently healthy that included 9 male and 9 female with age average (35-65 year) .The study revealed that telomere length(TL) were differences between males and females of control group. Age- adjusted telomere length were shorter in males than in females of control subjects (13,200 bp ) vs. (11,600 bp), (14,200 bp) vs. (13,000 bp) and (15,100 bp) vs. (13,800 bp) ,and this differences in TL between males and females decreased as aging increased, while this gender differences in TL was not observed among the diabetic patients .In both the controls group and diabetic subjects, the telomere length were shorter in older subjects than younger for both males and females. The result show differences in TL between control and diabetic subjects. TL were shorter in the patients with Type 2diabetes compared with the control subjects ,and TL in first duration of male diabetic patients(2,100 bp) in male(55 years) and (1,900 bp) in male (65 years) ,and second duration of female diabetic patients (3,600 bp) in female (55 years) and (2,900 bp) in female (65 years) were shorter compared with other duration. The differences in TL in patients with family history of disease shown shorter than patients without family history of disease and this differences in TL in patients with family history of disease from mother origin shorter than patients with family history of disease from father origin. The result of telomere length (TL) show that TL were shorter in males than females of control group while this differences absent in diabetic patients .TL was shorter in first duration of disease in males diabetic patients and in second duration of females diabetic patients compared with other duration of disease. TL was shorter in older subjects than younger for both groups and for both gender ,also shorter in diabetic patients from genetic origin of disease than non-genetic origin. Positive correlation between TL and estrogen in female control group and between  TL and adiponectin in diabetic patients was found. Keywords: Telomere, DNA, TR

Study the levels of adiponectin, FSH, LH and Sex hormones in Type 2 diabetes (NIDDM)

The hypothalamic/pituitary/gonadal (HPG)axis is central to the mammalian reproductive system . Pulsatile release of GnRH from neurons in the hypothalamus stimulates the secretion of LH and FSH from gonadotropes in the anterior pituitary. It has long been recognized that reproductive function is closely associated with energy balance, and metabolic dysregulation is linked with reproductive abnormalities (Lu et al.,2008). Compare the differences in levels of adiponectin, FSH, LH, testosterone and estradiol between the diabetic patients and control group and in diabetic patients according to the durations of disease for both males and females groups  .Also study the relationship between adiponectin and hormones for both gender and for both diabetic groups and control also in diabetic patients according to the durations of disease. About five milliliters of venous blood were collected from each subject in the study. The blood was separated by centrifugation at (3000 rpm) for 15 min. The sera were stored frozen at (-20 ºC) until assayed. This study was conducted between November 2010- November 2012 and, it was carried out at the diabetic Centre / Merjan Teaching Hospital in Babel Province, by taking 120 diabetic patients non- insulin dependent diabetes mellitus (NIDDM) (60 male  and 60 female)with disease durations(0-5),(>5-10 )and (>10 )years ,and with age average (35-65 year), most of them were on oral hypoglycemic drugs, and the study included 40 people apparently healthy that included  20 male and 20 female with age average (35-65 year) ,as control matched with disease groups. The statistical analysis of this study showed that patients with Type 2 diabetes of both males and females had significantly lower in adiponectin levels than control group (P<0.05),but this level of adiponectin significantly higher in females than males for both diabetic groups and control (p<0.05)  ,while the levels of FSH and LH had significantly higher in diabetes group than control (p<0.05) ,also significantly higher  in females than males for both diabetic groups and control (P< 0.05). The result of hormonal analysis show there were no significant differences in estradiol and testosterone between diabetic groups and control (p > 0.05). According to the durations of disease, the results show significant elevation in adiponectin level  in third duration of disease for both males and females groups as compared with first and second duration (P <0.004), while the levels of FSH, LH ,estradiol and testosterone show no significant differences among duration(P>0.05). According to the gender ,levels of adiponectin ,FSH, LH, and estradiol were significantly higher in females than males(P<0.05) , whereas levels of testosterone was significantly higher in males than females (P<0.05) . Correlation analysis showed an inverse correlation between adiponectin and FSH in female diabetic patients. Also an inverse correlation between adiponectin and estradiol was found in females of control group, while the correlation of LH and testosterone with adiponectin appears no significant correlation for both groups and for both males and females . According to the durations of disease, in first duration (0-5year)the study revealed positive correlation between adiponectin and FSH in male diabetic patients . In third duration (>10 year) an inverse correlation between adiponectin and FSH in female diabetic patients. The result also appears no significant correlation among adiponectin and LH, estradiol and testosterone in both gender among duration . The results of the present study indicate that the patients with Type 2 diabetes of both males and females had significantly lower in adiponectin levels than control group . levels of adiponectin significantly higher in females than males for both diabetic groups and control. Adiponectin levels significantly increased with duration of disease also with age.  Levels of FSH and LH had significantly higher in diabetic patients than control also significantly higher in females than males for both groups . Correlation analysis showed inverse correlation between adiponectin and both FSH and estradiol in female diabetic patients also inverse correlation between adiponectin and both FSH and estradiol in third duration of female diabetic patients

Study the relationship between adiponectin with thyroid hormones and cortisol in Type 2 diabetic patients (NIDDM)

Thyroid hormones has profound effects of lipid metabolism and carbohydrate homeostasis . Abnormalities in serum lipids and lipoproteins are frequent findings in thyroid dysfunction, mainly in hypothyroidism. Impaired glucose tolerance and insulin resistance have been documented in patients with thyroid dysfunction. In addition, thyroid hormones are remarkable regulators of energy metabolism, being the adipose tissue the largest fuel storage compartment . Furthermore, thyroid hormones share some physiological actions with adiponectin, such as reduction of body fat by increasing thermogenesis and lipid oxidation .However, a few number of studies have found low adiponectin levels in hypothyroid subjects (Díez and Iglesias, 2009). Cortisol excess in man is characterized by abdominal obesity, hypertension, glucose intolerance or diabetes and dyslipidemia. All these features share a state of insulin resistance , and contribute to high cardiovascular risk typical of this condition. Glucocorticoids negatively regulate adiponectin mRNA in human visceral adipose tissue. Cortisol counteracts the action of insulin at multiple sites, and increases hepatic gluconeogenetic efficiency (Fallo et al.,2004). Study the relationship between adiponectin with thyroid hormones and cortisol for both gender and for both control and diabetic groups also in diabetic patients according to duration of disease. This study was conducted between November 2010- November 2012 and, it was carried out at the diabetic Centre / Merjan Teaching Hospital in Babel Province by taking 120 diabetic patients(Type II DM) (60 male and 60 female) with disease duration (0-5),(>5-10 )and (>10 )years ,with age average (30-65 year)and most of them were on oral hypoglycemic drugs. While the study included 40 people apparently healthy that included 20 male and 20 female with age average (30-65 year) .About five milliliters of venous blood were collected from each subject in the study. The blood was separated by centrifugation at (3000 rpm) for 15 min. The sera were stored frozen at (-20 ºC) until hormonal assayed. Correlation analysis showed an inverse correlation between adiponectin and cortisol in female diabetic patients, while in male diabetic patients there was positive correlation between adiponectin and T4 was found. The correlation of TSH and T3 with adiponectin appears no significant correlation for both groups and for both males and females. The study revealed positive correlation between adiponectin and T4 in second and third duration in male diabetic patients , while an inverse correlation between adiponectin and TSH in male  diabetic patients in first duration. In female diabetic patients an inverse correlation between adiponectin and T3 in second duration,also found between adiponectin with cortisol in third duration of female diabetic patients. Correlation analysis showed an inverse correlation between adiponectin and cortisol in female diabetic patients, while positive correlation between adiponectin and T4 was found in male diabetic patients. This study show that Type 2 diabetic patients in first duration(0-5 year) an inverse correlation between adiponectin and TSH in male diabetic patients. In second duration(>5-10 year)an inverse correlation between adiponectin and T3 in female diabetic patients. In third duration(>10 year) an inverse correlation between adiponectin with cortisol  of female diabetic patients, while positive correlation between adiponectin and T4 in second and third duration of male diabetic patients

A systematic review of usability challenges and testing in mobile health

Nowadays, the combining of advanced mobile communications and mobile account now in portable devices named "smart phones" has becomes more great uses.Among of these include health care professionals. Few studies in the challenge, blurred reality challenge facing the patient and developer alike in the usability of mobile health.Therefore, this pape