Culicinae Mosquito TRA-2 RNA Interference Technique to Genetically Produce Maleness Population

Abstract

This invention entails a method to make a Tra-2 RNAi kernel sequence, and uses for this kernel sequence. The method includes the steps of amplifying an RNA recognition motive (RRM) DNA sequence from a Culcinae mosquito species, to obtain a first RRM DNA sequence of 240 base pairs; then reversely connecting the first RRM DNA sequence with a second RRM DNA sequence via an intron or a linker DNA sequence, in such a way that the second RRM DNA sequence forms an inverted sequence to the first RRM DNA sequence. Transcription of the two DNA sequences produces single strands of mRNA that can bind together to form a double strand hairpin mRNA structure. Other steps include inserting a tetracycline repressible transactivator, and an insect spermatogenesis promoter.

1 . A method to make a Tra-2 RNAi kernel sequence, which method comprises the steps of:
 i) amplifying an RNA recognition motive (RRM) DNA sequence from a  Culcinae  mosquito selected from the group consisting of  Aedes aldopictus, Aedus aegypti, Aedes polynesiensis  and  Culex quinquefasciatus,  to obtain a first RRM DNA sequence of 240 base pairs,   ii) repeating step i), to obtain a second RRM DNA sequence of 240 base pairs;   iii) reversely connecting the first RRM DNA sequence with the second RRM DNA sequence via an intron or a linker DNA sequence, which intron or linker DNA sequence is connected to the end of the first RRM DNA sequence and the beginning of the second RRM DNA sequence, so that the second RRM DNA sequence forms an inverted sequence to the first RRM DNA sequence,
 wherein the transcription of the first RRM DNA sequence and the second RRM DNA sequences produces single strands of mRNA with complementary sequences exposed at ends of the strands of mRNA, which complementary sequences are of sufficient length so as to be capable of binding together to form a double strand hairpin mRNA structure having a loop portion, where the loop portion is formed by the transcription product of the intron or linker DNA sequence. 
   
     
     
         2 . The method of  claim 1 , wherein the RRM DNA sequence is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3. 
     
     
         3 . The method of  claim 1 , wherein the intron or linker DNA sequence is connected to the end of the first RRM DNA sequence and the beginning of the second RRMA sequence via an AG sequence. 
     
     
         4 . A method to make a Tra-2 RNAi construct, which method comprises the steps of:
 i) creating an RNAi kernel sequence by
 amplifying an RNA recognition motive (RRM) DNA sequence from a  Culcinae  sequence selected from the group consisting of  Aedes aldopictus, Aedus aegypti, Aedes polynesiensis  and  Culex quinquefasciatus,  to obtain a first RRM DNA sequence of 240 base pairs, 
 amplifying an RNA recognition motive (RRM) DNA sequence from a  Culcinae  sequence selected from the group consisting of  Aedes aldopictus, Aedus aegypti, Aedes polynesiensis  and  Culex quinquefasciatus,  to obtain a second RRM DNA sequence of 240 base pairs, 
 reversely connecting the first RRM DNA sequence with the second RRM DNA sequence via an intron or a linker DNA sequence, which intron or linker DNA sequence is connected to the end of the first RRM DNA sequence and the beginning of the second RRM DNA sequence, so that the second RRM DNA sequence forms an inverted sequence to the first RRM DNA sequence, 
 wherein the transcription of the first RRM DNA sequence and the second RRM DNA sequences produces single strands of mRNA with complementary sequences exposed at ends of the strands of mRNA, which complementary sequences are of sufficient length so as to be capable of binding together to form a double strand hairpin mRNA structure having a loop portion, where the loop portion is formed by the transcription product of the intron or linker DNA sequence, 
   ii) inserting a tetracycline repressible transactivator operably linked to and controlling expression of the RNAi kernel sequence, and   iii) inserting an insect spermatogenesis promoter operably linked and controlling expression of the tetracycline repressible transactivator.   
     
     
         5 . The method of  claim 4 , wherein the RRM DNA sequence is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3. 
     
     
         6 . The method of  claim 4 , wherein the intron or linker DNA sequence is connected to the end of the first RRM DNA sequence and the beginning of the second RRMA sequence via an AG sequence. 
     
     
         7 . The method of  claim 4 , wherein the insect spermatogenesis promoter is  Drosophila -β2. 
     
     
         8 . The method of  claim 4 , which comprises the further step of inserting a regulatory component, or a terminator component, or both. 
     
     
         9 . The method of  claim 8 , wherein the regulatory component is tetOx7 and the terminator component is SV40 polyA. 
     
     
         10 . A method for increasing genetic maleness offspring in a  Culicinae  mosquito population, comprising the steps of: 
       producing a Tra-2 RNAi construct, which method comprises the steps of:
 a) creating an RNAi kernel sequence by
 i) amplifying an RNA recognition motive (RRM) DNA sequence from a  Culcinae  sequence selected from the group consisting of  Aedes aldopictus, Aedus aegypti, Aedes polynesiensis  and  Culex quinquefasciatus,  to obtain a first RRM DNA sequence of 240 base pairs, 
 ii) amplifying an RNA recognition motive (RRM) DNA sequence from a  Culcinae  sequence selected from the group consisting of  Aedes aldopictus, Aedus aegypti, Aedes polynesiensis  and  Culex quinquefasciatus,  to obtain a second RRM DNA sequence of 240 base pairs, 
 iii) reversely connecting the first RRM DNA sequence with the second RRM DNA sequence via an intron or a linker DNA sequence, which intron or linker DNA sequence is connected to the end of the first RRM DNA sequence and the beginning of the second RRM DNA sequence, so that the second RRM DNA sequence forms an inverted sequence to the first RRM DNA sequence, 
 wherein the transcription of the first RRM DNA sequence and the second RRM DNA sequences produces single strands of mRNA with complementary sequences exposed at ends of the strands of mRNA, which complementary sequences are of sufficient length so as to be capable of binding together to form a double strand hairpin mRNA structure having a loop portion, where the loop portion is formed by the transcription product of the intron or linker DNA sequence, 
 iv) inserting a tetracycline repressible transactivator operably linked to and controlling expression of the RNAi kernel sequence, and 
 v) inserting an insect spermatogenesis promoter operably linked and controlling expression of the tetracycline repressible transactivator, 
 
 b) stably transforming a  Culicinae  mosquito with the Tra-2 RNAi DNA construct, 
 c) allowing stable expression of the Tra-2 RNAi DNA construct during spermatogenesis in the  Culicinae  mosquito, so as to effect Tra-2 gene knockdown of X(m) chromosome-bearing sperm, 
 d) allowing the  Culicinae  mosquito of step ii) to mate and thereby stably pass on the Tra-2 RNAi DNA construct to offspring, resulting in continuous interruption of the development of X(m) chromosome-bearing sperm and thereby effecting genetic male bias in progeny. 
 
     
     
         11 . The method for increasing genetic maleness offspring in a  Culicinae  mosquito population of  claim 10 , wherein genetic male bias in progeny is at least 90%. 
     
     
         12 . A Tra-2 RNAi DNA construct produced by the method of  claim 4 . 
     
     
         13 . A  Culicinae  mosquito stably transformed with the Tra-2 RNAi DNA construct of  claim 12 , wherein expression of the DNA construct occurs during spermatogenesis so as to effect Tra-2 gene knockdown of X(m) chromosome-bearing sperms.