Genetic Transformation in Prokaryotic and Eukaryotic Cells

Abstract

Improving the quality and quantity of an organism and its products can be approached by molecular characters enhancement through the insertion of a gene of interest into cells of the desired organism. Genetic transformation of an organism involves isolation, identification, cloning a gene of interest into a vector, and transferring the gene to the target organism. This chapter reviews the process of genetic transformation into the organism’s cell from bacterial (Escherichia coli), yeast, plant (Onion, Tobacco, and Orchids), and mammalian. The discussion will be focused on the introduction of DNA molecules into plant cells and protoplast mediated by polyethylene glycol (PEG), electroporation, and gene gun using particle bombardment. Further discussion on the transient protein expression system of plant-based on protoplast, onion cell, and tobacco will also be covered in this chapter as well. The systems have been proven as a powerful tool for determining subcellular protein localization, protein-protein interactions, identifying gene function, and regulation. Finally, it can be clearly seen, the differences and similarities in the mechanism of genetic transformation both in prokaryotic and eukaryotic systems.

1. Introduction

To improve the quality and quantity of an organism, both prokaryotes and eukaryotes, it can be approached by molecular character enhancement through the insertion of interest genes or superior genes into the cells of the desired organism. The process of genetic transformation of an organism involves the isolation and identification of the gene of interest, the technique of cloning the gene on a plasmid vector until the process of transferring the gene to the target organism’s cell. One of the important genes in the growth of organisms is the homeobox gene, which is a gene that regulates the growth and development of organisms in a very early stage. Homeobox genes were first discovered in the Drosophila melanogaster. These homeobox genes have been also found in all multicellular organisms from fungi to plants, and vertebrate animals [1].

In plants, overexpression of the homeobox gene at an early stage of growth will activate the formation of apical buds from apical meristems that will produce shoots. The addition of exogenous cytokinin and auxin growth regulators will activate the homeobox genes to induce cell division genes that in turn will produce somatic embryos. Theoretically, each somatic cell can grow and transform itself into somatic embryos, therefore it can produce plant seeds in large quantities and uniform phenotypic characters. This is very profitable for agriculture and industry, especially for the mass production of identical plant seeds using tissue culture techniques.

In the model plant, Arabidopsis thaliana, it has been reported that the homeobox genes always maintain the growth of meristem cells in Shoot Apical Meristem (SAM) [2]. Overexpression of the homeobox gene in Arabidopsis has shown that the cells can convert from a determinate state to the meristematic indeterminate state, depending on the levels of expression of the gene (s) (Table 1) [23].

Gene	Function	Organism	Ref
OSH1	Homologous with Kn1 (Zea mays). Altered morphology of transgenic plants	Oryza sativa	[3, 4]
OSH15	Homologous with Kn1 (Zea mays). Affect the design of internodes resulting in stunted plants	Oryza sativa	[5]
KNAT1 and KNAT2	Morphogenesis	Arabidopsis thaliana	[6]
blr-1 and blr-2	Phyllotactic pattern and stem cell fate	Arabidopsis thaliana	[7]
ANL2	Accumulation of anthocyanin and in root development	Arabidopsis thaliana	[8]
ATHB-2	Mediates plant morphogenesis for light signals response	Arabidopsis thaliana	[9]
MDH1	Homologous BEL1 (Arabidopsis). Play an essential role in the control of plant fertility	Malus domestica Borkh	[10]
ATH1	Controls plant architecture by locally restricting environmental responses	Arabidopsis thaliana	[11]
GLABRA2	Effects seed oil content in Arabidopsis	Arabidopsis thaliana	[12]
WOX9	have species-specific roles in embryo and inflorescence development	Arabidopsis thaliana	[13]
WOX4	Regular TDIF Peptide Signaling Regulates Vascular Stem Cell Proliferation	Arabidopsis thaliana	[14]
LeT6	The leaves morphological states	Tomato	[15]
Athb-12	Response to treatment for abscisic acid (ABA)	Arabidopsis thaliana	[16]
KNAT6	Involved in Meristem Activity and Organ Separation	Arabidopsis thaliana	[17]
ATHB16	Regulates the photoperiod sensitivity in Arabidopsis and leaf development	Arabidopsis thaliana	[18]
ATK1	Transcriptional activator	Arabidopsis thaliana	[19]
ZmOCL1	Play a role in the embryo protoderm identity specification, organize of the primary root primordium or the L1 cell layer maintenance in the shoot apical meristem	Zea mays	[20]
BELL1	Master regulator for the gametophyte-to-sporophyte transition	Physcomitrella patens	[21]
Athb-2	Changes in light quality perceived by a novel phytochrome regulate plant development	Green plants	[22]
NTH15	Regulate leaf and flower morphology, accompanied by a decrease in the content of the active gibberellin		[14]

Table 1.

The homeobox gene in plants.

2. Transformation for transient expression in onion, tobacco leaves, and protoplast

Transient expression become a powerful tool in functional genomics study for detecting gene expression in a short time and the inserted gene do not integrate into the plant genome. A transient expression system has been developed in planta using different cells or tissues, including protoplast, onion cells, and tobacco (Nicotiana benthamiana) leaves (Table 2). A transient expression system using protoplasts has proven to be a good experimental tool in molecular biology. This approach is an efficient technique to study subcellular protein localization, protein complexes, in vivo gene silencing, and promotor activity [24, 25].

Plant materials	Transformation methods	Purposes/Functions	Refs.
Protoplast	PEG	Subcellular localization of proteins	[24, 25]
Nicotiana benthamiana leaves	Agroinfiltration	Metabolite’s production (protein, secondary metabolite, etc.)	[26]
Onion cells	Particle bombardment	Subcellular localization of proteins	[27]

Table 2.

Transient expression system and its purposes in planta.

The advantages of the transient expression system compared to stable expression are that it does not require regeneration of transformed cells, does not affect the stability of the host genome, and is independent of the effect of T-DNA integration site position [28]. Protoplast transfection can be performed using a variety of procedures commonly used for the transfection of animal cell cultures. The procedures that are often used to insert DNA into protoplasts are polyethylene glycol (PEG) and electroporation [29].

Polyethylene glycol (PEG)-mediated transformation plant cells can be transformed through certain chemicals, namely PEG (polyethylene glycol). PEG is an oligomer or hydrophilic polymer synthesized from ethylene oxide, containing repeating units of -(O-CH₂-CH₂)-. Polyethylene oxide (PEO) is another name for PEG. Typically, ethylene oxide macromolecules with a molecular weight of less than 20,000 g/mol are called PEGs, while macromolecules with values above 20,000 g/molar are called PEOs [29]. PEG is soluble in acetonitrile, benzene, water, ethanol, and dichloromethane, while it is insoluble in diethyl ether and hexane (Figures 1 and 2).

Figure 1.

Agroinfiltration in tobacco (Nicotiana benthamiana) leaves for protein-protein analysis.

Figure 2.

Transient expression in onion cell and protoplast for determining the subcellular localization of the protein. (a) Subcellular localization of OsKAN1-GFP fusion protein in the nucleus of onion cell transformed using particle bombardment [30]. (b) Transient expression of GFP-GF14c and Hd3a-mCherry in rice protoplast was driven by the 35S promoter of cauliflower mosaic virus and ubiquitin promoter, respectively, Bar = 10 μm.

PEG is available in various structures, such as branched, stellar, and comb-like macromolecules. PEG can bind various reactive functional groups to the PEG polymer site. Homo and heterobifunctional PEG derivatives are particularly suitable as agents or spacers of two chemical entities, whereas mono-functional PEGs prevent linking reactions that can affect the PEGylation of certain compounds with bifunctional PEGs. PEGylation is an interesting process in which PEG is bound to other molecules [31, 32].

PEG was used to increase DNA uptake into the protoplast during transfection. Very high concentrations of PEG can reduce transfection efficiency because it is toxic to protoplasts [33]. PEG-mediated DNA uptake is a direct gene transfer method that utilizes the interaction between PEG, naked DNA, salts, and protoplast membranes to influence the transport of DNA into the cytoplasm. The advantage of PEG-mediated transformation is that it does not require special equipment and can be carried out in the laboratory under sterile conditions [34]. Compared to Agrobacterium tumefaciens-mediated transformation, PEG-mediated transformation was not species-specific. In addition, PEG-mediated transformation is also useful for functional analysis of genes through transient expression, a technique that is often used for promoter analysis [35].

Particle bombardment particles are coated in DNA and can penetrate plant cells without killing the plant cells themselves. Previous experiments have shown that particle bombardment has been successfully used to insert DNA into rice callus and seedlings grown in dark conditions but has the disadvantage of low efficiency and reliance on expensive equipment [36].

3. Expression of recombinant psychrophilic RNase III in Escherichia coli

To understand the mechanism of how the transformation and expression of recombinant protein in a prokaryotic system, Escherichia coli BL21(DE3) have been used as host and recombinant RNaseIII as a model protein. Ribonuclease III is an enzyme that specifically cleaves the double-stranded RNA molecules. It functions for ribosomal RNA maturation; therefore, RNase III is indispensable for the survival of cells. Here, the production of recombinant psychrophilic RNase III from Shewanella sp. SIB1 in the Escherichia coli system was reported. As a psychrophilic enzyme, recombinant RNase III was produced in the form of inclusion bodies. To produce the soluble recombinant psychrophilic RNase III, co-expression with FKBP22 from the same bacteria was carried out. The result showed that FKBP22 significantly improved the solubility of recombinant psychrophilic RNase III. It strongly suggested that FKBP22 assists the proper folding of recombinant psychrophilic RNase III when it was overproduced in the Escherichia coli system.

Ribonuclease III (RNase III) is an enzyme that specifically cleaves double-stranded RNA [30, 37, 38, 39, 40]. RNase III has an important role in both the RNA transcript maturation and decay of diverse cellular and viral RNA. A primary function of RNase III, however, is the maturation of ribosomal RNA (rRNA) [30, 37, 38, 40, 41]. RNase III has been known to be widely distributed across the living kingdom of life, from bacteria to higher eukaryotes. RNase III family has common features in their molecular organization, by which it consists of catalytic domain with the common feature of HNERLFGDS located at the N-terminus and double-stranded binding domain (dsRBD) that located on their C-terminus [39]. RNase III exhibited enzymatically active in homodimeric form, by which each monomer has its catalytic mechanism and therefore the cleavage product of the RNase III exhibits a very regular length of short double-stranded RNA [39]. By such properties, RNase III can be manipulated to produce short dsRNA that can be implemented for the RNA interference technology in combination with Argonaute, Drosha, and Dicer [42]. Therefore, the production of recombinant RNase III is necessary from the scientific and technological point of view.

Production of recombinant proteins could be done in either bacterial or mammalian cells as a host. The choice of the host to produce recombinant protein may be the subject of proteins of interest. It depends on whether further processing of the proteins of interest is necessary or not. However, the bacterial cell is the most prominent host for recombinant protein production. Escherichia coli is the most common bacterial cell that is generally used as a host organism because of the following advantages—(a) it has unparalleled fast growth kinetics, (b) high cell density cultures are easily achieved, (c) the growth media are easily prepared and inexpensive, and (d) transformation with exogenous DNA is fast and easy [43]. There are several commercially available Escherichia coli appropriate for the expression host of recombinant proteins, such as Escherichia coli BL21(DE3) and its derivatives. Escherichia coli BL21(DE3) is carrying the T7gene1 from the lysogens DE3, a derivative of bacteriophage lambda, that encodes for T7 RNA polymerase under the control of lacUV5 promoter [44]. T7 RNA polymerase is a polymerase that can recognize T7 promoter, a strong promoter appropriate for the high-level expression of proteins. Such promoter is commonly used in several commercially available expression vectors, such as pET series, pRSET, and pACYC-Duet. These vectors contain a regulatory system in the form of lacI in which the gene product suppresses the expression of recombinant proteins.

This report will discuss the production of recombinant RNase III from a psychrotrophic bacterium, Shewanella sp. SIB1. Shewanella sp. SIB1 is a psychrotrophic bacterium that grows most rapidly at 20°C [45]. This strain can grow even at 0° but cannot grow higher than 30°C. Phylogenetic analysis indicates that Shewanella sp. SIB1 is closely related to the Shewanella sp. AC10 isolated from the Antarctic ocean [44]. Interestingly, protein from psychrotrophic bacterium exhibits distinct properties compared to the mesophilic counterparts by their ability to adapt to cold temperatures [45].

Protein adaptation in such low temperatures requires a strategy that is not commonly found in mesophilic, for example, psychrophilic proteins must be flexible enough to avoid the problem in protein folding and to perform the optimum catalytic activity if it is an enzyme. Therefore, the production of psychrophilic protein would be interesting due to their properties to adapt to such low temperatures. Although the production of recombinant protein in bacterial host seems to be straightforward, several difficulties that arise and how to solve the problems during the production of recombinant psychrophilic protein will be discussed.

3.1 Localization of Shewanella sp. SIB1 RNase III encoding gene (Sh-rnc)

To localize the Sh-rnc gene from the Shewanella sp. SIB1 genome, as well as to obtain the full length of the RNase III open reading frame, the inverse PCR was carried out in this work. Previously, the partial Sh-rnc gene was amplified by using a pair of primers constructed based on the sequence of open reading frames of the rnc gene from Shewanella oneidensis MR1. Once the fragment of the Sh-rnc gene was amplified then it was used to construct new primers for the inverse PCR. For the inverse PCR, the SIB1 genome was digested by the DraI restriction enzyme and then the digestion product was then allowed to perform self-ligation to form small circular products. Since the orf of the Sh-rnc gene contains a recognition site for DraI, therefore, the PCR was conducted by using two pairs of primers. By such a strategy, the two PCR products were obtained and were then cloned into pUC18 for sequencing. The sequencing results indicated that the two fragments corresponded to the lepB and era genes, which means that the rnc gene was flanked by the lepB and era genes at the upstream and downstream regions, respectively (Figure 3) [46]. It seems that the three genes are organized in one operon, since there was no promoter detected in the upstream of every orf of lepB, rnc, and era genes. The gene organization was similar to that of Rhodobacter capsulatus [41]. Based on the information of rnc gene organization in Shewanella sp. SIB1 genome, the full length of orf of rnc gene could be isolated and then used for the expression of recombinant psychrophilic RNase III. The length of the orf of the rnc gene was determined to be 678 bp, which produced the recombinant RNase III with a molecular weight of ±24.8 kDa.

Figure 3.

Molecular organization of rnc gene in Shewanella sp. SIB1 genome. The rnc gene is flanked by lepB and era genes at the upstream and downstream regions. It seems that lepB-rnc-era is organized in one operon since there was no promoter sequence was found at the upstream of each gene. Moreover, the rnc-era sequence overlaps with each other (hatched area), while lepB-era (white area) is separated only by one base. Arrows indicate the expression direction [46].

3.2 Expression of recombinant psychrophilic RNase III

To overexpress the recombinant psychrophilic RNase III from Shewanella sp. SIB1, the pET28a expression vector, and Escherichia coli BL21(DE3) as a host were used in this work. Insertion of the orf of rnc gene into the multiple cloning sites of pET28a produces the recombinant protein that is fused with the hexahistidine tag. The resultant plasmid, pET-rnc, was then used to transform Escherichia coli BL21(DE3). Expression of the recombinant psychrophilic RNase III was induced by isopropyl thio-b-D-galactopyranoside (IPTG).

The result showed that the recombinant psychrophilic RNase III was accumulated in inclusion body form, although the overproduction was shifted at 20°C (Figure 4). Several attempts have been implemented to improve the solubility of recombinant psychrophilic RNase III in the E. coli system. Shifting of the expression temperatures to 15 and 10°C and adjustment of pH of growing media also did not significantly improve the solubility of recombinant proteins (data not shown).

Figure 4.

SDS-PAGE of recombinant psychrophilic RNase III overproduced in Escherichia coli BL21(DE3). Samples were subjected to 15% SDS-PAGE and stained with Coomassie brilliant blue (CBB). Low molecular weight kit (GE Healthcare) (lane M); cell pellet of cell harboring pET-rnc without co-expression with FKBP22 (lane 1); soluble part of cell harboring pET-rnc without co-expression with FKBP (lane 2); cell pellet of cell harboring pET-rnc and FKBP22 (lane 3); and soluble part of cell harboring pET-rnc and FKBP22 (lane 4). Recombinant psychrophilic RNase III was indicated by arrow [46].

Another strategy that has been carried out to improve the recombinant psychrophilic RNase III was by co-expression with the chaperone or chaperone-like proteins. Chaperon is a protein that functions for assisting another protein folding. Two types of assisting folding proteins used were GroEL-ES from Escherichia coli and FKBP22 from Shewanella sp. SIB1 [47]. Among them, co-expression with FKBPP22 successfully improved the solubility of recombinant psychrophilic RNase III (Figure 2). FKBP22 belongs to the group of peptidyl-prolyl isomerase (PPIase) that functions for switching cis- to trans-configuration of proline during polypeptide biosynthesis [47]. This result indicated that strong induction to produce recombinant psychrophilic RNase III might cause the misfolding of the protein. Therefore, during co-expression with FKBP22, it helps to assist the proper folding of the psychrophilic RNase III. Although co-expression with FKBP22 only partly solubilizes the recombinant psychrophilic RNase III, it is sufficient for the biochemical characterization of the recombinant proteins.

Psychrophilic enzymes have unique properties in their folding and activity. Expression of such recombinant psychrophilic enzymes in mesophilic host generally produces misfolding recombinant protein represented by the inclusion bodies formation. Overexpression of recombinant psychrophilic RNase III in Escherichia coli has been improved when it was expressed with chaperone-like protein, FKBP22. It is apparently that FKBP22 assists the proper folding of recombinant psychrophilic RNase III.

4. Eukaryote model organism and animal gene transformation Yeast genetics

The yeast Saccharomyces cerevisiae is an essential option for expanding breakthrough research in gene cloning in E. coli, including eukaryotes. It can be manipulated and cultured using standard techniques applied to unicellular microorganisms. Yeast is a eukaryotes cell whose genetic material is packed into the chromosomes of the membrane-enclosed cell nucleus. In addition, extensive knowledge has been accumulated over the years that yeast has been used as a model system for genetic and biochemical studies. A comprehensive map showing the 17 chromosomes and more than 400 genes is available. The discovery first drove research in this area that yeast genes can be reliably expressed in E. coli. Yeast DNA fragments, when cloned into E. coli can restore histidine-independent growth of the mutant strain. In another case, a fragment of the yeast chromosome carries the gene for the enzyme that corresponds to the defect in the bacterial strain. Therefore, the yeast HIS3 gene can be expressed in bacterial cells and produce the yeast gene. Usually, wild-type alleles are specified in uppercase, and mutant ones are set in lowercase. Therefore, HIS3 is a wild-type allele, and his3 is a mutant allele that causes histidine dependence. Other yeast genes isolated and used as markers include TRP1, LEU2, URA3, and ARG4. In general, eukaryotic genes have more complex functions than bacterial genes due to introns. Due to the lack of introns, yeast genes may develop easier than other animal cells. An important marker of wild-type yeast attempts to insert exogenous DNA into yeast cells.

4.1 Yeast transformation

Yeast cells are protected by a thick cell wall, a potential barrier to DNA invasion. Removing the cell wall to create protoplasts or spheroplasts increases the chances of genetic transformation. Reseachers adopted this method was adopted and widespread used by these researchers, but some changes have since been have been reported to improve efficiency. This method is based on the technique described initially for protoplast fusion yeast. Yeast cells are recovered in the late stage of growth, the cell wall is weakened with a reducing agent such as mercaptoethanol, and the wall is removed by incubation with an enzyme such as glucanase. Various formulations, such as glucanase enzyme and actinomycete extract have been successfully used. Spheroplasts were then carefully washed with an osmotically equivalent solution of the free buffer and suspended in a solution-containing polyethylene glycol (PEG) and CaCl₂ [48]. DNA was added at this stage. For cells to divide, the walls need to be rebuilt. This case requires the cells to be placed in osmotically stabilized agar.

4.2 Gene recognition and gene number regulation

Both plasmid vectors and chromosomal integration are widely used to introduce genes and control copy numbers into S. cerevisiae. Each has an important role, and the choice depends on the overall goal (overexpression, tight control of gene number, etc.) [49]. The plasmids used in yeast are far more limited than the E. coli. However, plasmids with little copy number control and isolation stability can be a significant problem even in selective media. Homologous recombination is so efficient in S. cerevisiae that integrating genes into the genome provides an alternative and simple mechanism for introducing genes. Chromosomal integration also allows the insertion of several identical or different genes. It is critical for the gene expression of regulated metabolic pathways. There are classes of plasmids that replicate independently in yeast: YIp, YAC, YRp, Yep, and YCp [50, 51, 52, 53]. S. Sacevisiae has a multi-cloning site (MCS) for inserting expression cassettes. The YRp vector originates from replication such as Autonomously Replicating Sequence (ARS) without partition control. However, this plasmid is extremely unstable and is not widely used in metabolic engineering applications. In contrast, the widely used YCp and YEp vectors have been demonstrated in many applications. The YCp vector (centromere/CEN) has an origin of replication; the centromere sequence is maintained at 12 copies per cell and exhibits high isolation stability in selective media. Strong constitutive promoter expression can significantly affect plasmid stability, reduce average copy counts, and overwhelm intracellular metabolic pathways [54]. In extreme cases, the CEN/ARS vector provides overproduction. Due to the general lack of yeast plasmids, very high copy counts were maintained. On the other hand, defective marker promoters lead to increased copy counts [55]. Hundreds of copies have been reported on selective media, but this high copy count is not essential for survival [54]. Generally, such vectors help with the overexpression of product genes rather than metabolic engineering applications [49]. There are 11 classes of animals’ homeobox that share homology and function among yeast and animal (Table 3). Today, the use of model organisms to replace animal cells is increasing more rapidly due to animal-free thinking in social development. However, cloning and transformation in mammals remain important [51].

Class	Sub Class	Gene	Function	Organism	Ref
ANTP	EuHox	Hox1, Hox2, Hox3, Hox4, Hox5, Hox6–8, Hox9–15	Essential for normal T lymphocyte and activated natural killer cell function	Isodiametra pulchra (Xenacoelomorpha)	[55, 56]
	SuperHox	Evx, Meox, Mnx, En, Gbx, Ro, DLx, Nedx, Hex,	Essential for the development of the pancreas	Zebrafish	[55]
	ParaHox	Gsx, Pdx, Cdx	Regulate LGE patterning but oppositely control the balance between proliferation and differentiation in the neuronal progenitor pool.	Isodiametra pulchra (Xenacoelomorpha)	[55, 57]
	NK cluster	Msx, NK4, NK3, Lbx, Tlx, NK1, NK5, NK6, NK7	Essential for normal heart morphogenesis	Isodiametra pulchra (Xenacoelomorpha)	[55, 58]
		Emx, Hlx, Dbx, Barh1, Barx, Bsx, Bari, Vax, Noto, NK2.1, NK2.2, Msxlx, Abox	Regulate postnatal myogenesis, including muscle maintenance during aging and regeneration of acute and chronic muscle injury.	Isodiametra pulchra (Xenacoelomorpha)	[55, 59]
PRD	PAX	Arx, Alx, Hbn, Rax, Otp, Gsc, Otx, Pitx	Regulate the development of the animal olfactory system	Mouse	[55, 60]
		Pax 1/9, Pon, Pax2/5/8, Pax3/7, Pax4/6/10, Eyg, Pax-alpha,	Involved in the development of the eye structures of Drosophila	Drosophila melanogaster	[55, 61]
		Vsx, Dmbx, Drgx, Phox, Prop, Prrx, Repo, Shox, Unox, Hopx	Expressed in the presumptive midbrain at early developmental stages, and the hindbrain at later stages, with exclusion from the MHB	Ciona, Isodiametra pulchra (Xenacoelomorpha)	[55, 62]
LIM		Isl, Lmx, Lhx1/5, Lhx2/9, Lhx3/4, Lhx6/8,	Encoding gene regulation during the pituitary gland, eye, and pancreas, organs assembly that was presumably not present in the common ancestor of vertebrates.	Isodiametra pulchra (Xenacoelomorpha); C. elegans	[55]
TALE		Irx, Mkx, Pbx, Meis, Pknox, Tgif	Plays a critical role in tendon differentiation by regulating type I collagen production in tendon cells.	Isodiametra pulchra (Xenacoelomorpha)	[55, 63]
POU		Pou1, Pou2, Pou3, Pou4,	Essential for the active maintenance of the differentiated state of a neuron across phylogeny.	Isodiametra pulchra (Xenacoelomorpha)	[55, 64]
SINE		Six1/2, Six3/6, Six4/5,	Play an essential role in retinal development and influence that these proteins have on cell proliferation and growth	Isodiametra pulchra (Xenacoelomorpha)	[55, 65]
CUT		Cmp, Cux, Onecut	Developing kidney with expression restricted to the nephrogenic zone.	Isodiametra pulchra (Xenacoelomorpha)	[55, 66]
ZF		Zfhx, Zeb, Tshz	Specification of individual anterior neural precursors and promotes the expression of tph and synaptotagminB, required for the differentiation of serotonergic neurons.	Isodiametra pulchra (Xenacoelomorpha), sea urchin	[55, 67]
HNF		Hmbox, HNF	The inducible protective mechanism that inhibits LPS-induced ROS production and inflammation in EA.hy926 cells by the subsequent inhibition of redox-sensitive NF-κB and MAPK activation.	Isodiametra pulchra (Xenacoelomorpha)	[55, 68]
CERS		Cers	Synthesize ceramides containing mainly C20–C26 fatty acids, with little or no synthesis of C16- and C18-ceramides	Isodiametra pulchra (Xenacoelomorpha)	[55, 69]
PROSS		Prox	Expressed in the developing CNS, lens-secreting cone cells of the eye, and midgut. In the mouse, Prox 1 is expressed in many of the same tissues. Young neurons of the subventricular region of the CNS, developing eye lens, and pancreas. Expression is also detected in the developing liver and heart, as well as transiently in the skeletal muscles	Mouse, Drosophila melanogaster, Isodiametra pulchra (Xenacoelomorpha)	[55, 70]

Table 3.

Homeobox gene in animal.

4.3 Cloning in animal cells

The development of a vector system for gene transformation in animal cells is under consideration [71]. These vectors are required in biotechnology to synthesize recombinant proteins from genes that are not correctly expressed when cloned in E. coli or yeast. Human cloning techniques are sought after by clinical molecular biologists seeking to develop gene therapy techniques: Diseases are treated by introducing the cloned genes into patients [71]. The clinical aspect means that the most excellent attention is paid to the mammalian cloning system, but significant advances have also been made in insects. Cloning insects is fascinating because it uses a new type of vector that we have never encountered.

4.4 Cloning in mammals

Currently, gene cloning in mammals is performed for one of three reasons: (1) To produce recombinant proteins in mammalian cell culture and related farming techniques. Milk. (2) In gene therapy, human cells are manipulated to treat diseases. (3) Achieve gene knockout, an important technique used to determine the function of unknown genes. These experiments are usually performed on rodents, such as mice. Viruses as a mammalian clone vector have been known to be the key to cloning mammals for many years. The first cloning experiment with mammalian cells was performed in 1970 using a vector-based on Simian virus 40 (SV40) [72, 73]. The virus can infect several mammalian species following a lysogenic cycle in some hosts and others. SV40 has the same problem as e and has a calicivirus embedded in it. This is because packaging restrictions limit the amount of new DNA inserted into the genome. Therefore, cloning with the SV40 requires replacing one or more of the existing genes with DNA to clone. The original experiment replaced the late gene region segment, but early gene replacement was also an option [73]. However, the discovery of CRISPR/Cas which is based on cloning technology is one of the essential techniques in gene therapy [74].

5. Conclusions

Genes are the universal language that controls the nature of all living things, shared homology among organisms. It is always interesting to reveal the evolution of cloning and gene expression in plant, bacteria, and animal cells. Therefore, with the discovery of genetic engineering, possible to exchange good genetic traits which beneficial for human life. In conclusion, genetic transformation is a genetic engineering technique that can be used to understand the function of a gene or several genes in various events in the life of an organism, both prokaryotes and eukaryotes, so that genetic transformation is carried out for two kinds of purposes, namely scientific purposes to determine the function of certain genes in an organism, and economic goals to improve the quality and productivity of an organism to increase the economic value of an organism. In the future, genetic engineering on prokaryotes and eukaryotes perspective can be used for various purposes in the fields of medicine, agriculture, horticulture, forestry, and food.

Acknowledgments

We thank Badan Penerbit dan Publikasi Universitas Gadjah Mada (BPP UGM) for supporting this publication.

Conflict of interest

The authors declare no conflict of interest.

Appendices and nomenclature

35S

Cauliflower Mozaic Virus 35S promoter

Argonaute

a part of RNA-induced silencing complex (RISC), plays a central role in RNA silencing processes

BIP116b

brassinosteroid Interacting protein 116b

Dicer

human RNase III

Drosha

a class III of RNase III

dsRBD

double-strand binding domain

dsRNA

double-strand RNA

era

era protein-encoding gene

FKBP22

peptidyl-prolyl isomerase protein, a chaperone-like protein from psychrophilic bacterium Shewanella sp. SIB1

GFP

green fluorescent protein

GroEL-ES

chaperonin

Hd3a

heading date 3a

IPTG

isopropyl thio-b-D-galactopyranoside

lepB

signal peptidase encoding gene

OsKANADI

Oryza sativa KANADI

P19

RNA silencing suppressor p19

pACYC-Duet

bacterial expression vector

pET

bacterial expression vector

pRSET

bacterial expression vector

RNase III

ribonuclease III

Rnc

ribonuclease III encoding gene

SDS-PAGE

sodium dodecyl sulfate-polyacrylamide gel electrophoresis

Sh-rnc

riboniclease III encoding gene from Shewanella sp. SIB1

T-DNA

transfer DNA

ANTP

antennapedia

ARG4

argininosuccinate lyase

Arx

aristaless related homeobox

Alx

aristaless-like homeobox

Hbn

homeobrain

Rax

retina and anterior neural fold homeobox

Otp

orthopedia homeobox

Gsc

goosecoid homeobox

Otx

orthodenticle homolog

Pitx

paired-like homeodomain

CaCl₂

calcium chloride

CERS

ceramide synthase

Cmp

collagen-mimetic peptide

Cux

cut-like homeobox

Onecut

one cut homeobox

CUT

cut homeobox

Emx

empty spiracles homeobox

Hlx

H2.0-like homeobox

Dbx

developing brain homeobox

Barh1

BarH-like 1 homeobox protein