Myostatin Transgenic Mice

8/3/2019 Myostatin Transgenic Mice

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Expression of Myostatin ProDomain Results In MuscularTransgenic Mice

Arena Ahmad

Hana Albaghoai

Fatin Iffah Rasyiqah binti Mohamad Zoolkefli

Nur Syuhada bt Alwi


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OBJECTIVES

To down-regulate the expression of

myostatin in order to enhance the

development of muscle and decrease the

fat content (increase in muscle mass)

To create the healthier livestock animals

with the increase in quality of meatproduction


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Introduction

Myostatin is a genetic determinant of skeletal muscle mass in

bovine and mice.

Also known as GDF-8, belongs to the TGF- superfamily.

TGF- will become activate in dimer state due to associationwith N-terminal propeptides in the forms of latent TGF-

complexes.

TGF- family members will undergo mutation and truncation

in order to decrease level process of myostatin and increase

the growth and development of muscle mass.

- Pro-domain TGF-1 cDNA was able to form latent

complexes with mature TGF-1, inhibit biological activity


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This down-regulation of myostatin can promote the growth of

muscle (increase the meat production for livestock animals,

e.g. pigs)

Down regulation of gene function can be done by :

- reduce myostatin RNA

- block myostatin protein production

- interfere the myostatin function

There are 2 to approach in order to down-regulate the

function the myostatin gene which are

-ribozyme based construction

-pro-domain construction


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Transgeneconstruction

Pro-domain Ribozyme

Microinjection &

detection

Detection of transgeneexpression

Analytical method

-Growth and carcass evaluation

-Determination of muscle fibresize and number

- Statistical analysis

METHODOLOGY


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Ribozyme-based transgene

Ribozymes (or RNA enzymes) are catalytic RNA capable ofcleaving target RNA molecules in a sequence-specific manner. Theribozyme transgene can be obtained from the secondary structure

of mouse myostatin mRNA.

Ribozyme-based transgenes are an effective way of reducingtarget RNA in tissue culture but had a checkered record in

transgenic animals. They have been successfully used in reductionof glucokinase and b2-microglobulin mRNA in transgenic mice.


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Pro-domain Transgene

Pro domain is a segment of the myostatin

protein and the coding sequence can beobtained by amplification PCR of mousemyostatin cDNA .


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Trangenic Construct of MLC-rib and MLC-pro


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pMEX-NMCS2vector,

ribozyme multimeror pro domainDNA sequence

Multiple clonningsite


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Method of Producing Transgenic

Mice This construct cloned into pMEX

NMCS2 vector. The vector is digested

with Not 1 restriction enzyme, theninserted into mouse genome using thepronuclear microinjection technique


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The Expression of The Myostatin InTransgenic Mice (MLC-rib and MLC-

pro)

Expression of the ribozyme - muscle developmentwas not altered.

However, a dramatic muscling phenotype wasobserved in transgenic mice carrying the myostatinpro domain gene and result in:

22-44% increase in muscle mass

17-30% faster growth rate compared to the non-transgenic control mice.


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3 transgenic lines (HIGH, MEDIUM, LOW)No other detectable RNA in other tissue

Transgene expression in the MLCpro mice


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Carcass Analysis

Was not performed in MLC-Rib micebecause of there were in difference inbody weight. Since the differencedetected in MLC-pro mice the carcassanalysis were preformed


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Fig. 4 Phenotype of transgenic and littermate control mice from HIGH expressing line.Transgenic mice and littermate control are shown on left and right respectively. (A) liveanimals, (B) whole carcasses, (C) fore limbs , (D) hind limbs.


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Transgenic mice which having HIGH expressing line,appeared larger and exhibit pronounced muscling inhips and shoulders.

However, during analysis of carcasses it can be seenthat the presence of the MLC-pro transgeneexpression is differed between male and female.


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HIGH and MEDIUM expressing line- Increase in weight of whole carcass, trunk,fore limbs, and hind limbs.

LOW expressing line

- Did not differ from control (Non-transgenicmice).


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FIGURE 5(Live and carcass weight of transgenic and sex-matched

littermate control mice at week 6 and 18)


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FIGURE 6(Weights of carcass components from transgenic and sex-

matched littermate control mice at 6 and 18 weeks)


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In males, the levels of expression of transgene and

degree of exhibited phenotype is directly linearrelationship which showed similar in increase.

In females, the levels of expression of transgene andmuscular phenotype relationship is not dramatic as inmales and become significant in all three levels oftransgene expression in 18-week.


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FIGURE 7(Effects of the MLC-pro transgene on muscle

fibre)


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Collectively, the increased musclemass by this transgene most likely

resulted from increased fibre cross-sectional size (hypertrophy).


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Effects of MLC Pro Transgene onEpididymal Fat , Heart and OtherOrgans To determine if the transgene influenced body fat deposition. Levels of transgene expression did not influence epididymal

fat, heart and other organs.

Some of the organs weights were reduced in comparison tocontrol animals.

The transgene increased live body and carcass weight butdid not result in increased internal organ in relation to theirlarger body size.


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Effects MLC Pro Transgene onreproduction

The MLC Pro transgenic mice from all lines were healthy, andable to reproduce well without fertility or litter- deliveryproblems.

The transgenic mice did not have any problems duringdelivery despite increase in body size.


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Summary

Muscle growth can be enhanced by inhibiting myostatin, a

negative regulator of muscle growth.

In this study, developed transgenic mice in which the

myostatin was suppressed by overexpression of myostatin

protein known as the prodomain

Expression of the propeptide in transgenic mice showed a22-44% increase in muscle mass and 17-30% fastergrowth rate compared to the non-transgenic control mice.


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contd

Transgenic mice did not display the muscularphenotype until one month of age, and theyappeared normal, healthy, and withoutreproduction problems.

This mouse model suggests that a transgenicanimal based on myostatin pro domain approachcan be used to develop farm animals withenhanced growth performance.

Increased musule mass & decreased fat contentwhich would equate to a healthier meat productfor consummers

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Myostatin Transgenic Mice

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