Navigating gene editing in porcine embryos: Methods, challenges, and future perspectives.

The term "transgenic animal" refers to an animal whose genome has been intentionally altered, as opposed to resulting from a spontaneous mutation. Transgenic animal technologies are among the rapidly advancing techniques in biotechnology. These technologies are used to insert exogenous genes into an animal's genome through genetic engineering methods, enabling the genes to be inherited and expressed by the animal's offspring. Several methods are available for creating transgenic animals, including microinjection, somatic cell nuclear transfer, retrovirus-mediated gene transfer, sperm-mediated gene transfer, testis-mediated gene transfer, and embryonic stem cell-mediated techniques. Transgenic technology allows for the transfer or enhancement of beneficial nutritional traits. For instance, increasing the omega-3 fatty acid content in fish could potentially lower the risk of coronary heart disease. Transgenic pigs have been created with higher levels of omega-3 fatty acids, and the alpha-lactalbumin gene has been inserted into cows to boost the human protein content in their milk (2.4g/liter). A transgenic animal is one that carries foreign DNA deliberately incorporated into its genome for specific scientific objectives. These animals can be engineered to improve human welfare across sectors like agriculture, industry, and medicine. While research on transgenic animals offers numerous benefits and holds promise for advancements in various scientific fields, the primary aim of this technology is to enhance traits such as milk production, wool quality, weight, disease resistance, feed efficiency, and reproductive performance. Transchromosomal animals may also be used for producing human therapeutic polyclonal antibodies. The use of transgenic animals presents potential solutions in drug research, xenotransplantation, disease resistance, and tissue repair. This review highlights different transgenesis methods and their diverse applications. Transgenic animals represent a key technology for the future, offering significant benefits to society.Top of FormBottom of Form

Metal–organic frameworks (MOFs) are porous materials with unique characteristics that make them well-suited for drug delivery and gene therapy applications. Among the MOFs, zeolitic imidazolate framework-8 (ZIF-8) has emerged as a promising candidate for delivering exogenous DNA into cells. However, the potential of ZIF-8 as a vector for sperm-mediated gene transfer (SMGT) has not yet been thoroughly explored.This investigation aimed to explore the potential of ZIF-8 as a vector for enhancing genetic transfer and transgenesis rates by delivering exogenous DNA into sperm cells. To test this hypothesis, we employed ZIF-8 to deliver a plasmid expressing green fluorescent protein (GFP) into mouse sperm cells and evaluated the efficiency of DNA uptake. Our findings demonstrate that ZIF-8 can efficiently load and deliver exogenous DNA into mouse sperm cells, increasing GFP expression in vitro. These results suggest that ZIF-8 is a valuable tool for enhancing genetic transfer in SMGT, with important implications for developing genetically modified animals for research and commercial purposes. Additionally, our study highlights the potential of ZIF-8 as a novel class of vectors for gene delivery in reproductive biology.Overall, our study provides a foundation for further research into using ZIF-8 and other MOFs as gene delivery systems in reproductive biology and underscores the potential of these materials as promising vectors for gene therapy and drug delivery.

The use of poultry as a unique model of biological research was characterised by a high level of efficiency, however, methods for creating transgenic ducks, complicated by the structure of waterfowl eggshells, are of low efficiency. The purpose of the study was to determine the influence of various biotechnological procedures for creating transgenic ducks on their productive qualities and reproductive ability to identify the optimal method for creating transgenic poultry for further use in scientific, research, or economic purposes. Weighting, morphometric and statistical analysis of productive traits were used during the study. 40 ducks (4 experimental groups of animals and about 3,000 of their eggs) were studied. The lowest value of the egg productivity index was obtained in the group created by busulfan injection (79.5±11.8%), the highest – in the group created by sperm-mediated gene transfer (91.8±2.3%), the group of direct injection of transgenic construct – 89.0±2.0%, which indicates that this biotechnological method of introducing transgenic construct did not have a clear effect on this indicator. The weight of ducks in different experimental groups ranged from 1,323.50±65.36 g (using the sperm-mediated gene transfer) to 1,608.08±94.76 g (in the group created using busulfan). Ducks that received direct injections had an average weight of 1,480.42±35.01 g. In the control group, the average weight at sexual maturity was 139.5±9.67 g, in the busulfan group – 148.2±13.13 g, in the direct injection group – 143.16±7.25 g, and in the spermmediated gene transfer group – 140.67±13.13 g. It was found that the method of injection into the embryo of a recipient sterilised with busulfan and the introduction of donor blastodermal cells negatively affect the reproductive qualities of ducks. The practical significance of the study lies in the fact that as a result of the analysis of the productivity of ducks obtained by various methods of transgenesis, it was determined that the most effective of the evaluated methods is the transfection of DNA of the transgenic construct with sperm (Sperm-mediated gene transfer, SMGT)

The advent of nanotechnology since the 1950s, when the well-known physicist Richard P. Feynman talked in his famous talk about "There's plenty of room at the bottom", has led to incredible contribution of nanotechnology in the fields of medical and veterinary therapeutics, diagnostics and other applications. Semen biology dealing with the study of spermatozoa and its related physiological and pathological aspects has not remained unscathed from the facets of nanotechnology. With each passing day investigators are revealing newer aspects of the nanoparticles, such as an antioxidants to relieve oxidative stress during semen cryopreservation, for the depletion of moribund spermatozoa from semen, gender selection of spermatozoa, bio-imaging of gametes, sperm mediated gene transfer, as well as for male fertility evaluation. As, the uses of various magnetic nanoparticles in the industry have gained acceleration, the evaluation of their effects, either beneficial or otherwise on the mammalian spermatozoa becomes obligatory. Many toxicological studies have also been conducted in respect to the harmful effects of different metallic nanoparticles related to their applicability, and industry borne adverse effects on the male germ cells in human beings and the animals. This review has been designed to focus on the beneficial as well as toxicological effects of various metallic nanoparticles on the mammalian spermatozoa and the future prospects related to their applicability in the semen biology.

Exogenous DNA length and quantity affect the transfection rate, but not sperm viability during Sperm-Mediated Gene Transfer

The present study was aimed to investigate isolation, cloning and se-quencing of chicken Growth hormone gene (cGH) from chicken (cobb 500 broilers) then transfer chicken Growth hormone gene by two methods the first gene transfer method by using sperm-mediated gene transfer (SMGT) technique and the second gene transfer method using bioresonance (Bio) to local chicken strain (Bandarah).This study was investigate for two generation. The body weight at hatch and 4 weeks of age in-creased by 3.32 and 8.09 g for SMGT method and by 1.27 and 5.22g for Bio method respectively. The SMGT method was increased the body weight at 12 wk of age by 60.17 and 7.96 g for males and females respectively. The growth rate during 0-4 weeks of age was 131.64, 130.68 and 132.35%, for SMGT, Bio and control respectively. In conclusion cGH Gene successfully isolate, molecular cloning from Cobb 500 and transferring by two methods SMGT and Bio to produce transgenic chickens of a local strain in Egypt .SMGT is an efficient method that will hopefully facilitate the imple-mentation of strategies for securing the benefits that can be expected to arise from the introduction of transgenic chicken, Bio open important new perspectives in the field of animal transgenic would be more rapid, with quick and effective delivery of genes to target tissues. Chicken cGH gene was effect in all growth traits and moved from the first generation to the second generation with the same shape and increased the effect. The second generation gave higher body weight at 0,4,8 and 12 weeks of age.

The experimental was aimed to improve genetic of Bandarah Local chicken strain to insert Growth hormone gene (cGH) from (Cobb 500) broiler strain by method sperm-mediated gene transfer technique (SMGT). This study investigated for two generations. Total RNA was extracted from chicken liver tissue and the cDNA was successfully prepared. PCR Products GH, mRNA normal Length 810bp for transgenic Bandarah chickens which treated by SMGT method, the same result was found in the first and second generation. The averages of fertility percentage were 88.97% and 92.14 % for SMGT and control, respectively. The overall mean of the second generation was nearly value 90.76% of the first one 90.38%.The responses of the transgenic techniques SMGT for fertility percentage were 5.64%. The responses of hatchability for SMGT were 18.35% and 21.82% for fertile and total eggs, respectively The SMGT of transferring was decreased the age at sexual maturity ASM at the second generation by 35.01 d. Egg number which produced during the first 90 days from SMGT method significantly increased compared by the control. There was highly significant difference between treated found, the SMGT method had the heaviest egg weight 49.20 g followed by control one 48.22 g. After two generations, the SMGT technique was improved egg number by 10.62 egg, decreased egg weight by 0.9g and improved egg mass during the first 90 days of laying by 512.62 g. SMGT is an efficient method that will hopefully facilitate the implementation of strategies for securing the benefits that can be expected to arise from the introduction of transgenic chicken. Chicken cGH gene was affected in egg productive traits and reproductive traits and moved from the first generation to the second generation with the same shape and increased the effect. Growth hormone gene transfer by SMGT will save time to improve egg productive traits and reproductive traits.

Goat (Capra hircus) stearoyl-CoA desaturase 1 (SCD1) plays a crucial role in fatty acid metabolism including milk and muscular fatty acid. This study investigated the expression of SCD1 gene in goat and examined its inheritability and expression in transgenic SCD1 mice and goats. Our results suggested the possibility of generating transgenic goats by sperm-mediated gene transfer (SMGT). The expression of SCD1 gene and fatty acid metabolism genes in goat mammary gland tissues and muscular tissues was aligned with GEO database, and tested by qRT-PCR. F 1 transgenic mice were mated and then the target genes and protein expression level in F 2 transgenic mice, as well as fat content in F 1 and F 2 transgenic mice muscle were examined. Successful transgenic goats generation was identified by testicular injection. The results showed a developmental time specific SCD1 expression in the goat mammary gland tissue and muscular tissue. The rate of positive expression of exogenic gene and exogenic protein in F2 transgenic mice was 30% and 6.67%, respectively, Moreover, muscular fat content in the positive group was significantly higher than the control group both in F1 and F2 (P > 0.05). The rate of positive expression of exogenic gene and exogenic protein was 10.29% and 8.82% in the transgenic goat by testicular injection. Results of the present study showed that SCD1 gene plays a crucial function in goat fatty acid metabolism, and that SCD1 can regulate fat synthesis in SCD1-transferred mice and be inherited stably. Also, SCD1transferred goat could be generated through testicular injection, suggesting a practical approach for transgenic livestock breeding.

Capillary electroporation affects the expression of miRNA-122-5p from bull sperm cells.

Sperm-mediated gene transfer (SMGT) has a potential use for zebrafish transgenesis. However, transfection into fish sperm cells still needs to be improved. The objective was to demonstrate the feasibility of tip type electroporation in zebrafish sperm, showing a protocol that provide high transfection efficiency, with minimal side-effects. Sperm was transfected with a Cy3-labelled DNA using tip type electroporation with voltages ranging from 500 to 1500 V. Sperm kinetics parameters were assessed using Computer Assisted Semen Analysis (CASA) and cell integrity, reactive oxygen species (ROS), mitochondrial functionality and transfection rate were evaluated by flow cytometry. The transfection rates were positively affected by tip type electroporation, reaching 64.9% ± 3.6 in the lowest voltage used (500 V) and 86.6% ± 1.9 in the highest (1500 V). The percentage of overall motile sperm in the electrotransfected samples was found to decrease with increasing field strength (P < 0.05). Increase in the sperm damaged plasma membrane was observed with increasing field strength (P < 0.05). ROS and sperm mitochondrial functionality did not present a negative response after the electroporation (P > 0.05). Overall results indicate that tip type electroporation enhances the internalization of exogenous DNA into zebrafish sperm cells with minimal harmful effects to sperm cells.Electronic supplementary materialThe online version of this article (10.1007/s11033-020-05658-2) contains supplementary material, which is available to authorized users.

To edit the duck genome by HDR-directed integration of the EGFP gene into the duck host genome in combination with SMGT using CRISPR/Cas9. Methods. HDR-mediated gene of green fluorescent protein (EGFP) was crried out by the combined action of four plasmids. The pX330 contained the Cas9 gene. Two plasmids contained sgRNA genes: pBR322-sgRNA1 and pBR322-sgRNA2. The pBR322-HDR-EGFP plasmid was constructed to contain the DNA vector with left homologous sequence part(LHP), the EGFP gene coding sequences and the right homologous sequence part(RHP). The DNA sequence data for designing the HDR-EGFPinsert and sgRNA 1 and sgRNA 2 were taken from the genome DNA sequence of Anas platyrhynchos Spindlin 1 (SPIN1) gene. Twenty four ducks (13 males and 11 females) of the Shaoxing breed were used for this experiment. The sperm transfection was performed using Lipofectamine 2000. Results. Thirty one ducks were obtained, 19 of which carried the EGFP gene. F2 analysis revealed that 16 ducks (F1) (14 females and 2 males) transmitted the transgene DNA to their offsprings. Thus 27.6 % (56/203) of F2 descendants were positive for the transgene DNA construct. Conclusions. Exogenous DNA was successfully inserted into the duck genome.

The ability to introduce controlled modifications of the genome of animals represents an important tool for biomedical and veterinary research. Among transgenic techniques, we describe here the sperm-mediated gene transfer method that is based on the spontaneous ability of sperm cells to bind and internalize exogenous DNA and to carry it to theoocyte during fertilization, producing genetically modified animals.

For years, extensive efforts have been made to use mammalian sperm as the mediator to generate genetically modified animals; however, the strategy of sperm-mediated gene transfer (SMGT) is unable to produce stable and diversified modifications in descendants. Recently, haploid embryonic stem cells (haESCs) have been successfully derived from haploid embryos carrying the genome of highly specialized gametes, and can stably maintain haploidy (through periodic cell sorting based on DNA quantity) and both self-renewal and pluripotency in long-term cell culture. In particular, haESCs derived from androgenetic haploid blastocysts (AG-haESCs), carrying only the sperm genome, can support the generation of live mice (semi-cloned, SC mice) through oocyte injection. Remarkably, after removal of the imprinted control regions H19-DMR (differentially methylated region of DNA) and IG-DMR in AG-haESCs, the double knockout (DKO)-AG-haESCs can stably produce SC animals with high efficiency, and so can serve as a sperm equivalent. Importantly, DKO-AG-haESCs can be used for multiple rounds of gene modifications in vitro, followed by efficient generation of live and fertile mice with the expected genetic traits. Thus, DKO-AG-haESCs (referred to as 'artificial spermatids') combed with CRISPR-Cas technology can be used as the genetically tractable fertilization agent, to efficiently create genetically modified offspring, and is a versatile genetic tool for in vivo analyses of gene function.

Naked DNA has been shown to bind naturally to the sperm, a method called sperm-mediated gene transfer (SMGT). Based on these observations, we examined the efficiency of exogenous DNA binding to sperm using liposomes. In this experiment, we analyzed methods to select frozen-thawed bovine sperm, and evaluated the binding of exogenous DNA to those sperm. To determine the optimal selection method, we used Computer-Assisted Sperm Analysis (CASA). Percoll or Swim-Up were used to select sperm, followed by incubation up to 3h with the liposome-DNA complexes. The samples were collected after 1h and after 3h. We used enhanced green fluorescent protein (eGFP) in combination with the liposomes as a marker for exogenous DNA binding. Five treatments per selection method were analyzed: (1) no incubation, no liposomes and no DNA, (2) incubation with no liposomes and no DNA, (3) incubation with liposomes and no DNA, (4) incubation with liposomes and 1µg of DNA and (5) incubation with liposomes and 10µg of DNA. The CASA results for total motility and rapid motility were statistically significant (P < 0.01) between the control and the other treatments in the Percoll group as opposed to Swim-Up. Swim-Up was therefore chosen as the optimal selection method. In order to determine if the liposome-DNA complex had bound to sperm, real time PCR was used to detect GFP DNA and images of the sperm were analyzed using the Spatial Light Interference Microscopy (SLIM). SLIM confirmed the presence of liposomes on the sperm head and tail.

Sperm-mediated gene transfer (SMGT) is based on the ability of spermatozoa to bind exoge- nous DNA and transfer it into oocytes by fertilization. However, SMGT is still undergoing opti- mization to improve its efficiency to produce transgenic animals. The acrosome reaction is neces- sary for spermatozoa to carry the exogenous DNA into oocytes. In this study, the effect of the acrosome reaction on the efficiency of spermatozoa carrying exogenous DNA was evalua- ted. The results showed that the efficiency of the acrosome reaction was significantly higher (p⟨0.05) after incubation with 50 μmol/L progesterone compared to incubation without proges- terone. It was significantly higher (p⟨0.05) in the 20, 40, and 60 min of progesterone treatment groups than in the 0 min treatment group. The spermatozoa were further incubated with cyanine dye Cy5 labeled DNA (Cy5-DNA) for 30 min at 37°C, and positive fluorescence signals were detected after the acrosome reaction was induced by progesterone at concentrations of 0 and 50 μmol/L for 40 min. The percentage of positive Cy5-DNA signals in spermatozoa was 96.61±2.06% and 97.51±2.03% following exposure to 0 and 50 μmol/L progesterone, respective- ly. The percentage of partial spermatozoa heads observed following combination with Cy5-DNA was 39.73±3.03% and 56.88±3.12% following exposure to 0 and 50 μmol/L progesterone, respec- tively. The ratio of positively stained spermatozoa combined with exogenous DNA showed no reduction after the acrosome reaction. These results suggest that the acrosome reaction might not be the key factor affecting the efficiency of SMGT.

The binding of exogenous DNA to sperm is a key process for sperm-mediated gene transfer; however, the underlying molecular mechanisms have yet to be elucidated. In the present study, we aimed to identify the DNA binding proteins (DBPs) in rabbit sperm and to gain further understanding of the molecular mechanism of sperm and exogenous DNA interaction. Native polyacrylamide gel electrophoresis was used for separating free sperm proteins and complexes of DNA fragment/sperm proteins. A distinct band was found after Coomassie blue staining, and seven potential proteins were identified by mass spectrometry analysis. An analysis of the physical/chemical properties of the seven proteins revealed that the sperm inner acrosomal membrane protein IAM38 (IAM38) matched the features of the DBPs. Western blotting analysis showed that the IAM38 and CD4 were present in the sperm but not in the seminal plasma. Blocking of the IAM38 impaired the DNA-binding capacity of the sperm. Blocking the CD4 decreased the DNA-uptake capacity of the sperm but did not influence the DNA-binding capacity of the sperm. Moreover, the EGFP-positive embryos and EGFP-positive blastocysts were also decreased after IAM38 blocking or CD4 blocking in comparison with the control group. In conclusion, our results imply that foreign DNA first binds to the transmembrane IAM38 of the sperm plasma membrane and then forms the complex of DNA/IAM38/CD4 with CD4 to complete the transportation of exogenous DNA into the nucleus of sperm.

SummaryOne of the methods to generate transgenic animals is called sperm-mediated gene transfer (SMGT). Mature sperm cells can take up exogenous DNA molecules intrinsically and transfer them into the oocyte during fertilization. This study assessed the effect of dimethyl sulfoxide (DMSO) and electrolyte-free medium (EFM) on DNA uptake (EGFP-N1plasmid) in mouse sperm. Sperms cells cultured in human tubular fluid (HTF) without any treatment were considered as the control group. Sperms cells that were incubated in EFM and HTF with DNA/DMSO at 4°C were classified into EFM and HTF groups. Sperm motility and viability were assessed following treatment. In vitro fertilization (IVF) with sperm in all groups was performed. Fertilization, embryo development and GFP-positive blastocyst rates were analyzed and compared. The result showed that sperm motility and viability in EFM were better than those in the HTF group. The rate of development to reach the blastocyst stage and GFP-positive blastocysts was significantly higher in the EFM group compared with the HTF group (P<0.05). Our data demonstrate that sperm stored in the EFM group can improve the efficiency of SMGT for the generation of GFP-positive blastocysts.

Abstract. Sperm-mediated gene transfer (SMGT) has been considered as an innovative device for transgenesis on a mass scale by taking advantage of live spermatozoa to transfer exogenous DNA. However, the fertilizing ability of transfected sperm cells and the poor reproducibility of this method are still matters of controversy. Hence, the current study was conducted to evaluate transfecting the enhanced green fluorescent protein (EGFP) as the source of exogenous DNA into bovine spermatozoa using a high-performance polymer reagent as well as assessing the fertilizing capacity of transfected sperm cells by in vitro fertilization (IVF). In the first experiment, three different concentrations of rhodamine-labeled DNA and high-performance polymer transfection reagent, X-tremeGENE HP, were used to transfect bovine spermatozoa. In the second experiment, IVF and fluorescence microscopy methods were utilized to assess the fertilizing capacity of sperm cells carrying exogenous DNA when X-tremeGENE HP was used either alone or with dimethyl sulfoxide (DMSO) treatment. Findings revealed that at 1 µL X-tremeGENE HP and 1 µg of DNA concentration, approximately one-third of total spermatozoa were transfected. However, following IVF and fluorescence microscopy, no EGFP expression was detected in zygotes and morula-stage embryos. Results of this study showed that, although X-tremeGENE HP could transfer EGFP to bovine spermatozoa, transfected sperm cells were unable to transfer foreign DNA to matured bovine oocytes. Under our experimental conditions, we hypothesized that the absence of the EGFP fluorescence signal in embryos could be due to the detrimental effects of transfection treatments on sperm cells' fertility performance as well as incompetency of IVF to produce transgenic embryos using transfected sperm cells.

As an effective method of transgenesis, the plasmid of PiggyBac transposon containing GFP (PiggyBac) transposon system has been widely used in various organisms but not yet in mollusks. In this work, piggyBac containing green fluorescent protein (GFP) was transferred into the Pacific oyster (Crassostrea gigas) by sperm-mediated gene transfer with or without electroporation. Fluorescent larvae were then observed and isolated under an inverted fluorescence microscope, and insertion of piggyBac was tested by polymerase chain reaction (PCR) using genomic DNA as template. Oyster larvae with green fluorescence were observed after transgenesis with or without electroporation, but electroporation increased the efficiency of sperm-mediated transgenesis. Subsequently, the recombinant piggyBac plasmid containing gGH (piggyBac-gGH) containing GFP and a growth hormone gene from orange-spotted grouper (gGH) was transferred into oysters using sperm mediation with electroporation, and fluorescent larvae were observed and isolated. The insertion of piggyBac-gGH was tested by PCR and genome walking analysis. PCR analysis indicated that piggyBac-gGH was transferred into oyster larvae; genome walking analysis further showed the detailed location where piggyBac-gGH was inserted in the oyster genome. This is the first time that piggyBac transposon-mediated transgenesis has been applied in mollusks.