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  Section: General Biotechnology / Animal Biotechnology
 
 
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Manipulation of Reproduction and Transgenic Animals

 
     
 

Content

Manipulation of reproduction in animals

 

Artificial insemination

    Semen collection and its storage

    Ovulation control

    Sperm sexing

  Embryo transfer

    Multiple ovulation (superovulation)   

    Multiple ovulation with embryo transfer

    Embryo splitting

    Embryo sexing

  In vitro fertilization (IVF) technology

 

 

In vitro Maturation (IVM) of oocytes

 

 

Culture of in vitro fertilized embryos

  Embryo Cloning

    Quadriparental hybrid

    Nuclear transplantation (Dolly)

    Embryonic stem cells

In vitro fertilization and embryo transfer in humans

  Infertilities in humans

    Male sterility

    Female sterility

  Who benefits IVF

  How the patients for IVF treated

    Indicators of ovary stimulation

    Oocyte recovery and uptake

    Semen preparation

  IVF and embryo transfer

Transgenic animals

  Strategies for gene transfer

    Transfer of animal cells/embryo

    Treatment through microinjection

    Targeted gene transfer

  Transgenic animals

  Transgenic sheep

  Transgenic fish

  Animal bioreactor and molecular farming

Application of molecular genetics

  Selected traits and their breeding into livestock

    Diagnosis, elimination and breeding strategies of genetic diseases

  Application of molecular genetics in improvement of livestock

    Hybridization based markers

    PCR-based markers

    Properties of molecu­lar markers

    Application of molecular markers

    Transgenic breeding strategies



Application of Molecular Genetics
Methods have been developed to isolate animal genes and to characterize them. The first approach is to identify DNA sequence associated with economic trait loci. The second step is to incorporate the identified gene into three maps : (i) a physical map of chromosome (where DNA sequences are assigned to specific sites on specific chromosomes), (ii) a linkage map (where linkage of different genes and markers is assigned in the same chromosome), (iii) a genetic map (where inheritance of economic traits is corrected with the inheritance of genes and markers).

The unknown and novel genes associated with genetic diseases can also be identified through their expression. Then the protein sequences are identified through amino acid sequencing or injecting the protein into suitable animal (e.g. mouse, rabbit, chicken, etc) to raise the antibodies.

The animal production industry is gradually reaching towards revolution due to the development of detailed linkage and genetic map, more knowledge of expression and regulation of genes and techniques for large propagation. The application of molecular genetics in animals currently being used are : breeding selected traits into livestock, animal cell culture, and production of transgenic animals.

Selected Traits and Their Breeding into Livestocks
There are several genetic traits known which can be bred into livestock if required, for example disease resistant traits. Some traits are determined from the coordinated expression of many genes but not single gene. The discipline of quantitative genetics unravels the total number of genes contributing to a trait, contribution of individual trait and its location on the chromosomes. Altogether they are termed as quantitative trait loci (QTLs), therefore, herds are surveyed for reference animals that have been bred for a broad range of phenotypic variations.

Diagnosis, elimination and breeding strategies of genetic diseases
An increase in homozygosity of recessive genes and the frequency of specific alleles at QTLs has been resulted due to isolation of domestic animals in small breeding group. Consequently most breeds of livestocks comprises of variant genes which are harmful or lethal at homozygous stage (when it contain two variant genes). Such allele may be found in 15 per cent cases with 5 per cent homozygotes. Several variant genes responsible for genetic diseases have been identified. Diagnostic tests are available which permit animals as normal, carrier (i.e. heterozygous with one normal allele or one variant allele) or affected (i.e. homozygous with two variant alleles). Now PCR is used to amplify the region of genes which has been affected. Thus amplified DNA fragement is used to identify the variants.

It is well known fact that genetic disease occurs when a gene malfunctions and enzyme is not expressed. When both alleles are defective in homozygote these results in phenotypic defects. The carriers (heterozygote) are phenotypically normal. Therefore, the genetic defects can be diagnosed by genetic tests and rectified biotechnologically during pregnancies that affect foetus (for detail see Genetic Engineering for Human Welfare).

 

The second approach for elimination of defect from a herd is the use of normal (homozygous) animals for mating. Use of MOET with embryo biopsy and splitting on the best animals is the other method of spread of genetic disease and curing them. Pre-identified carrier or affected embryos should not be transferred (this aspect has been discussed earlier) (Read and Smith, 1996).

Application of Molecular Markers in Improvement of Livestock

During the last two decades the progress in recombinant DNA technology and gene cloning has brought in revolutionary changes in the field of basic as well as applied genetics. Several new approaches for genome analysis have been made. Now it is possible to uncover a large number of genetic polymorphism at the DNA sequence level and to use them as markers for evolution of genetic basis for the observed phenotypic variability (Mitra et al. 1999).


Usually a marker is considered as a constituent that determines the function of construction. Variations occurring at different levels i.e. at the morphological, chromosomal, biochemical or DNA level can serve as genetic markers. Thus genetic markers can be defined as any stable and inheritable variation that can be measured or detected by a suitable method, and that can be used to detect the presence of a specific genotype or phenotype other than itself. The markers revealing variations at DNA level are referred to as 'molecular markers'. So far an unlimited number of molecular markers is known since the first demonstration of DNA level polymorphism in 1974, which is called restriction fragment length polymorphism (RFLP). The molecular markers are classified into two broad categories, the hybridization-based markers and the PCR-based markers.

The hybridization-based markers
This includes the traditional RFLP analysis. During RFLP analysis well labeled probes for important genes (e.g. cDNA or genomic sequence) are hybridized onto filter membrane containing restriction enzyme digested DNA. Then these are separated by gel electrophoresis and subsequently transferred onto these filters by Southern blotting. Thereafter, the polymorphisms are observed as hybridization bands. The individuals that carry different allelic variants for a locus will show different banding pattern. Hybridization can also be carried out with the probes (e.g. genomic or synthetic oligonucleotide) for the different families of hypervariable repetitive DNA sequences such as minisatellite, simple repeats, variable number of tandem repeats (VNTR) and microsatellite to reveal highly polymorphic DNA fingerprinting pattern (Mitra et al, 1999).

The PCR-based markers
There is no need of probe-hybridization step. The PCR-based markers have lead to discovery of several useful methods which are easy to screen. On the basis of types of primers (i.e. primers of specific sequences targeted to particular region of genome or primers of arbitrary sequences) used for PCR. These markers further can be subdivided into two groups, the sequence-targeted PCR assay, and the arbitrary PCR assay.

(i) The sequence-targeted PCR assay. In this assay system, a particular fragment of interest is amplified using a pair of sequence-specific primers. In this category, PCR-RFLP or cleaved amplified polymorphic sequence (CAPS) analysis is a useful technique for screening of sequence variations that give rise to the polymorphic restriction enzyme (RE) sites. A specific region of DNA encompassing the polymorphic RE sites is amplified. The amplified DNA fragment is digested with respective RE. The variations in sequence are screened by several approaches.

(ii) The arbitrary PCR assay. In this assay randomly designed primer is used to amplify a set of anonymous polymorphic DNA fragments. When primer is short, there is high probability of priming taking place at several sites in genome that are located within amplifiable distance and are in inverted orientation. Polymorphism detected by using this method 'is called randomly amplified polymorphic DNA (RAPD). Based on this principle several techniques have been developed which differ in number and length of primer used, stringency of PCR conditions and the method of fragment separation and detection.

Properties of molecular markers
In genetic analysis, many types of markers viz., morphological, chromosomal, biochemical and molecular markers are used. Morphological (e.g. pigmentation and other features) and chromosomal (e.g. structural and numerical variations) markers usually show low degree of polymorphism, therefore, they are not very useful. Biochemical markers have been tried out extensively but have not been found encouraging as they are sex linked, age dependent and influenced by the environment. The molecular markers capable of detecting the genetic variations at the DNA sequence level, have removed the limitations. They possess unique genetic properties that make them more successful than the genetic markers. They are numerous, distributed on genome, follow typical Mendalian inheritance and are multiallelic giving heterozygosity of more than 70 per cent and unaffected by environmental factors.

For genetic analysis, molecular markers offer several advantages. Mitra et al. (1999) have discussed the several advantages of molecular markers: (i) the DNA samples can easily be isolated from blood, tissues e.g. sperms, hair follicle as well as archival preparations, (ii) the DNA samples can be stored for a longer time and readily be exchanged between the laboratories, (iii) the analysis of DNA can be carried out at an early age or even at the embryonic stage, irrespective of sex, (iv) once the DNA is transferred onto a solid support e.g. filter membrane, it can be repeatedly hybridized with the different probes and heterologous probes and in vitro synthesized oligonucleotide probes can also be used, and (v) the PCR-based methods can be subjected to automation (Mitra et al., 1999).

Application of molecular markers
Polymorphism observed at the DNA sequence level has been playing a major role in human genetics for gene mapping, pre- and post- natal diagnosis of genetic diseases, and anthropological and molecular evolution studies. Similar approach for exploitation of DNA polymorphism as genetic markers in the field of animal genetics and breeding has opened vistas in livestock improvement programmes. Much interest has been generated in determining variability at the DNA sequence level of different livestock species, and in their assessment whether these variations can be exploited efficiently in conventional as well as transgenic breeding programmes.

\Molecular markers can play important role in livestock improvement through conventional breeding strategies. The various possible applications of molecular markers are: short-range (immediate) applications and the long-range applications (Table 7.1).

Table 7.1. Molecular markers useful in conventional livestock breeding.

 

 

Application

Marker system

1.

Short-range/immediate Application

 

 

 

Parentage determination

DFP, microsatellite

 

 

Genetic distance estimation

DFP, RAPD, microsatellite

 

 

Determination of zygosity / Freemartinism

RFLP, PCR-RFLP, microsatellite RFLP,

 

 

Sex determination

PCR-RFLP, DFP, microsatellite

 

 

Identification of disease carrier

RFLP, CAPS, microsatellite

2.

Long-range Application

 

 

 

Gene mapping

Type II markers e.g. VNTR, minisatellite,  microsatellite, RAPD

 

 

Marker-assisted selection

Any marker having direct/indirect association with the performance traits/QTL under question

Source: Mitra et al. (1999).; RAPD, randomly amplified polymorphic DNA; CAPS, cleaved amplified polymorphic sequence; DFP, DNA fingerprinting; QTL, quantitative trait loci.

Transgenic breeding strategies
The current breeding strategies of livestock largely rely on the principle of selective breeding. In this method genetic improvement is brought about by increasing the frequency of advantageous alleles of many loci. The actual loci are rarely identified. In these methods genes cannot be moved from distant sources like different species or genera due to reproductive barrier.

The recent development in molecular biology has given rise to new technology called transgenesis, which has removed the breeding barriers between different species or genera. Transgenesis has opened up many vistas in understanding behavior and expression of a gene. It has made possible to alter the gene structure and modify its function (Rusconi, 1991). There are many applications of transgenesis, but the most convincing one is the development of transgenic dairy animals for the production of pharmaceutical proteins in milk, and animals with altered milk composition (Wall et al, 1997).

The transgenesis first starts with identification of the genes of interest. In this context, molecular markers can serve as reference point for mapping relevant genes. After successful production of transgenic animals, appropriate breeding methods could be followed for multiplication of transgenic herd. Molecular markers can also be used for identification of the animals carrying the transgenes. Most of the QTL are polygenic in nature and transgenesis presently single gene traits are being manipulated (Wall et aL, 1997). The technology holds future promises in moving polygenic QTL across the breeding barriers of animals. However, it is expected that molecular markers wili serve as a potential tool to geneticists and breeders to evaluate the existing germplasm, and to manipulate it to create animals of desired traits as demanded by the society (Mitra et al, 1999).

 
     
 
 
     



     
 
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